Recombinant endotoxin-neutralizing proteins

ABSTRACT

In general, the invention features a recombinant endotoxin-neutralizing polypeptide (RENP) characterized by (i) an amino acid sequence, (ii) an amino acid sequence and structure that facilitates selective and specific binding to lipopolysaccharide and (iii) once bound to the lipopolysaccharide, provides endotoxin-neutralizing activity. Preferably, the RENP is composed of an amino acid sequence similar to, but not identical to, an amino acid sequence of BPI, LBP, or both. Preferably, the RENP contains an LPS-binding domain derived from the amino acid sequence of BPI, LBP, or both. Preferably, the RENPs are covalently bound to a molecule which enhances the half-life of the polypeptide. The RENPs of the invention can be used in pharmaceutical compositions for therapeutic and prophylactic regimens, as well as in various in vitro and in vivo diagnostic methods.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of:

[0002] 1) U.S. application Ser. No. 07/915,720, filed Jul. 22, 1992,which is the U.S. national phase application of PCT Internationalapplication no. PCT/US91/05758, filed Aug. 13, 1991, which was filed inthe PCT designating the U.S. as a continuation-in-part of U.S.application Ser. No. 07/681,551, filed Apr. 5, 1991, now U.S. Pat. No.5,171,739, issued Dec. 15, 1992, which is a continuation-in-part of U.S.application Ser. No. 07/567,016, filed Aug. 13, 1990, now abandoned,which is a continuation-in-part of U.S. application Ser. No. 07/468,696,filed Jan. 22, 1990, now U.S. Pat. No. 5,089,274, issued Feb. 18, 1992,which is a continuation-in-part of U.S. application Ser. No. 07/310,842,filed Feb. 14, 1989, now abandoned; and

[0003] 2) PCT International application no. PCT/US94/04709, filed Apr.29, 1994, which was filed in the PCT designating the U.S. as acontinuation-in-part of U.S. application Ser. No. 08/165,717, filed Dec.10, 1993, which is a continuation-in-part of U.S. patent applicationSer. No. 08/056,292, filed Apr. 30, 1993, which is acontinuation-in-part of U.S. application Ser. No. 07/567,016, filed Aug.13, 1990, now abandoned, which is a continuation-in-part of U.S.application Ser. No. 07/468,696, filed Jan. 22, 1990, now U.S. Pat. No.5,089,274, issued Feb. 18, 1992, which is a continuation-in-part of U.S.application Ser. No. 07/310,842, filed Feb. 14, 1989, now abandoned.These applications are each incorporated herein by reference and areapplications to which we claim priority under 35 U.S.C. §120 or §365(c).

FIELD OF THE INVENTION

[0004] This invention relates generally to the field of recombinant,endotoxin-neutralizing proteins, particularly to recombinant proteinswhich bind endotoxin and block endotoxin-mediated activation ofbiological systems.

BACKGROUND OF THE INVENTION

[0005] Gram-negative infections are a major cause of morbidity andmortality, especially in hospitalized and immunocompromised patients.[Duma, Am. J. of Med., 78 (Suppl. 6A):154-164 (1985); and Kreger et al.,Am. J. Med., 68:344-355 (1980)]. Although available antibiotics aregenerally effective in inhibiting growth of Gram-negative bacteria, theydo not neutralize the pathophysiological effects associated withendotoxins. Endotoxin is a heat stable bacterial toxin composed oflipopolysaccharides (LPS) released from the outer membrane ofGram-negative bacteria upon lysis [Shenep et al., J. Infect. Dis.,150(3):380-388 (1984)], and is a potent stimulator of the inflammatoryresponse. Endotoxemia occurs when endotoxin enters the bloodstreamresulting in a dramatic systemic inflammatory response.

[0006] Many detrimental in vivo effects of LPS result from solublemediators released by inflammatory cells. [Morrison et al., Am. J.Pathol., 93(2):527-617 (1978)]. Monocytes and neutrophils, which ingestand kill microorganisms, play a key role in this process. Monocytes andneutrophils respond to endotoxin in vivo by releasing soluble proteinswith microbicidal, proteolytic, opsonic, pyrogenic,complement-activating and tissue-damaging effects. These factors mediatemany of the pathophysiological effects of endotoxin. For example, tumornecrosis factor (TNF), a cytokine released by endotoxin-stimulatedmonocytes, causes fever, shock, and alterations in glucose metabolismand is a potent stimulator of neutrophils. Other cytokines such as IL-1,IL-6, and IL-8 also mediate many of the pathophysiologic effects of LPS,as well as other pathways involving endothelial cell activation bytissue factor, kininogen, nitric oxide and complement.

[0007] Endotoxin-associated disorders result from extra-gastrointestinalexposure to LPS, e.g. administration of LPS-contaminated fluids, orGram-negative infections. Endotoxin-associated disorders can also resultwhen the natural epithelial barrier is injured and the normalGram-negative flora breach this barrier. For example,endotoxin-associated disorders can occur (a) when there is ischemia ofthe gastrointestinal tract (e.g., following hemorrhagic shock or duringcertain surgical procedures), or (b) when systemic or local inflammationcauses increased permeability of the gut to endotoxin or Gram-negativeorganisms. The presence of endotoxin and the resulting inflammatoryresponse may result, for example, in endotoxemia, systemic inflammatoryresponse syndrome (SIRS), sepsis syndrome, septic shock, disseminatedintravascular coagulation (DIC), adult respiratory distress syndrome(ARDS), cardiac dysfunction, organ failure, liver failure (hepatobiliarydysfunction), brain failure (CNS dysfunction), renal failure,multi-organ failure and shock.

[0008] Examples of diseases which can be associated with Gram-negativebacterial infections or endotoxemia include bacterial meningitis,neonatal sepsis, cystic fibrosis, inflammatory bowel disease and livercirrhosis, Gram-negative pneumonia, Gram-negative abdominal abscess,hemorrhagic shock and disseminated intravascular coagulation. Subjectswho are leukopenic or neutropenic, including subjects treated withchemotherapy or immunocompromised subjects (for example with AIDS), areparticularly susceptible to bacterial infection and the subsequenteffects of endotoxin.

[0009] Several therapeutic compounds have been developed to inhibit thetoxic effects of endotoxin, including antibacterial LPS-binding agentsand anti-LPS antibodies, although each has met with limitations. Forexample, Polymyxin B (PMB) is a basic polypeptide antibiotic which bindsto Lipid A, the most toxic and biologically active component ofendotoxin. PMB inhibits endotoxin-mediated activation of neutrophilgranule release in vitro and is a potential therapeutic agent forGram-negative infections. However, because of its systemic toxicity,this antibiotic has limited therapeutic use, and is generally usedtopically. Combination therapy using antibiotics and high doses ofmethylprednisolone sodium succinate (MPSS) showed more promise as thisregimen prevented death in an experimental animal model of Gram-negativesepsis. However, a clinical study using MPSS with antibiotics intreatment of patients having clinical signs of systemic sepsis showedthat mortality rates were not significantly different between thetreatment and placebo groups [Bone et al., N. Engl. J. Med. 317:653(1987)].

[0010] Antibodies that bind endotoxin have been used in the treatment ofendotoxemia. For example, hyperimmune human antisera against E. coli J5reduced mortality by 50% in patients with Gram-negative bacteremia andshock [Ziegler et al., N. Engl. J. Med. 307:1225 (1982)]. However,attempts to treat Gram-negative sepsis by administration of anti-LPSmonoclonal antibodies met with little or no success [Ziegler et al., N.Engl. J. Med. 324:429 (1991); Greenman et al., JAMA 266:1097 (1991);Baumgartner et al., N. Engl. J. Med. 325:279 (1991)].

[0011] Another approach to treating endotoxemia involves the use ofcytokine blockers, such as IL-1 receptor antagonists and anti-TNFantibodies, as well as the soluble forms of the IL-1 and TNF receptors.However, any given cytokine blocker blocks only the cytokine for whichit is specific, and fails to prevent the action of other cytokines.Furthermore, blocking cytokines may have other deleterious effects.

[0012] Two soluble endotoxin-binding proteins, lipopolysaccharidebinding protein (LBP) and bactericidal/permeability-increasing (BPI),play opposing roles in vivo in the physiological response to endotoxin.LBP is a soluble LPS receptor found in serum which binds LPS with highaffinity via interaction with the Lipid A moiety [Tobias et al. (1986)J. Exp. Med. 164:777-793; to Tobias et al. (1989) J. Biol. Chem.264:10867-10871]. LBP-LPS complexes stimulate monocyte activationthrough interaction with the CD14 receptor on the surface of monocytes,resulting in production of cytokines such as TNF and IL-1 [Wright et al.(1989) J. Exp. Med. 170:1231-1241; Wright et al. (1990) Science249:1431]. Thus, LBP acts as a transfer protein in LPS-mediatedstimulation of cytokine release. Moreover, LBP increases LPS activity inthat a lower concentration of LPS is required to stimulate monocytes inthe presence of LBP than in its absence.

[0013] In direct contrast to LBP, BPI binds and neutralizes endotoxin,preventing inflammatory cell activation. BPI, also known as CAP57 and BP[Shafer et al., Infect. Immun. 45:29 (1984); Hovde et al., Infect.Immun. 54:142 (1986)] is also bactericidal by virtue of its interactionwith the Lipid A moiety of LPS in the bacterial cell wall. BPI bindsLPS, disrupts LPS structure and the cell wall, and increases bacterialmembrane permeability, resulting in cell death [Weiss et al., J. Biol.Chem, 253:2664-2672 (1978); Weiss et al., Infection and Immunity38:1149-1153 (1982)]. BPI retains its in vitro bactericidal activityafter protease cleavage, suggesting that BPI fragments retain activity[Ooi et al., Clinical Research 33(2):567A (1985)]. This observation wasconfirmed by Ooi et al., who showed that an N-terminal 25 kD fragment ofBPI exhibited both the in vitro bactericidal and permeability increasingactivities [Ooi et al., J. Biol. Chem. 262:14891 (1987)].

[0014] Molecular Structures of BPI and LBP

[0015] The genes encoding BPI and LBP have been cloned [Gray et al.(1989) J. Biol. Chem. 264:9505-9509; Schumann et al., Science249:1429-1431 (1990)]. BPI and LBP are immunologically cross-reactive,contain a hydrophobic leader sequence, and share significant amino acidsequence to homology over the entire length of the molecules, with anoverall amino acid sequence identity of 44% [Tobias et al., J. Biol.Chem. 263:13479-13481 (1988); Schumann et al. supra]. BPI and LBP eachcontains three cysteine residues. BPI contains two glycosylation sites;LBP contains five potential glycosylation sites.

[0016] BPI is characterized by two distinct domains, an N-terminaldomain and a C-terminal domain, which are separated by a proline-richhinge region. The N-terminal domain of BPI has strong LPS-neutralizingactivity, while the C-terminal domain of BPI has modest LPS-neutralizingactivity. LBP can also be divided into N- and C-terminal domains, withthe C-terminal domain being implicated in binding of LPS to macrophagesand their subsequent activation.

[0017] The N- and C-terminal domains of BPI have a striking chargeasymmetry that is not shared by LBP. The N-terminal domain of BPI, whichis rich in positively charged lysine residues, imparts a predicted pI>10to the full-length molecule. In contrast, the C-terminal domain of BPIis only slightly negatively charged. LBP, which is a neutral protein,has no bactericidal activity [Tobias et al., J. Biol. Chem. 263:13479(1988)]. This suggests that the bactericidal activity of BPI resultsfrom its overall cationicity.

[0018] Table 1 provides a comparison of BPI and LBP structure andfunction. TABLE 1 Comparison of BPI and LBP Structure and Function BPILBP SYNTHESIS Site of synthesis Neutrophil Liver Blood concentration1-10 ng/ml 1-10 μg/ml STRUCTURE Molecular mass 55 kD 60 kD Glycosylationsites 2 5 Cysteine 3 3 EFFECTS ON LPS MEDIATED: neutrophil activationInhibits Stimulates monocyte activation Inhibits Stimulates TNF releaseInhibits Stimulates IL-1 release Inhibits Stimulates IL-6 releaseInhibits Stimulates

[0019] Therapeutic intervention to block the inflammatory effects of LPSwould ameliorate the morbidity and mortality associated with endotoxemiaand septic shock. Unfortunately, although BPI binds LPS with highaffinity, it has an extremely short half-life in vivo, thus limiting itsuse in therapy. Native LBP has a longer half-life but, upon binding ofLPS, elicits a brisk monocyte reaction which can facilitate release ofdeleterious quantities of cytokines.

[0020] Early and specific diagnosis of endotoxin-associated disorders isessential in the identification of patients who have or who are at riskof developing such disorders.

[0021] Precise identification of a site of Gram-negative infection in apatient would assist the clinician in the design and targeting ofantibacterial therapy.

[0022] An ideal anti-endotoxin drug candidate and/or LPS detectionreagent would have a longer half-life and effective, high-affinityendotoxin binding/inactivation without monocyte stimulation. There is aclear need in the field for specific diagnostic and therapeutic agentswhich neutralizes the effects of endotoxin and has an acceptably longhalf-life in vivo. The present invention addresses these problems.

SUMMARY OF THE INVENTION

[0023] Recombinant proteins are genetically engineered to bindlipopolysaccharide (LPS) such that the endotoxin is inactivated, thuspreventing the endotoxin from inducing the immunological cascade ofevents associated with endotoxin-related disorders (e.g., activation ofmonocytes, tumor necrosis factor (TNF) production).

[0024] In general, the invention features a recombinantendotoxin-neutralizing polypeptide (RENP) characterized by (i) an aminoacid sequence, (ii) a sequence and structure that facilitate specificbinding to lipopolysaccharide, (iii) provides endotoxin-neutralizingactivity upon LPS binding, and (iv) a half-life that is enhancedrelative to the half-life of BPI. Preferably, the RENP is composed of anamino acid sequence similar to, but not identical to, an amino acidsequence of BPI, LBP, or both. Preferably, the RENP contains anLPS-binding domain derived from the amino acid sequence of BPI, LBP, orboth. Preferred RENPs are fusion proteins which bind LPS with the highaffinity of BPI, but do not contain the BPI amino acid sequencesassociated with BPI's short half-life.

[0025] Preferably, the RENPs are covalently bound to a molecule whichfurther enhances the half-life of the polypeptide. For example, thehalf-life enhancing molecule can be an immunoglobulin fragment, ahalf-life determining portion of LBP or LBP derivative, or polyethyleneglycol. In related aspects, the invention features DNA encoding an RENPof the invention, vectors and transformed cells containing DNA encodingan RENP, a method for producing RENPs, and detectably labeled RENPs.

[0026] A primary object of the invention is to provide an RENP whichbinds and inactivates endotoxin, and has a half-life suitable foradministration to a patient.

[0027] Another object of the invention is to provide a pharmaceuticalcomposition containing a therapeutically effective amount of an RENP foruse in treatment of endotoxin-related disorders.

[0028] Still another object of the invention is to provideendotoxin-neutralizing proteins for use in the detection of LPS. TheRENPs can be bound to a label which can be detected or can be bound to asupport for use in LPS-detection assays. LPS can be detected in vivo toidentify a site of infection in a subject or can be used in an in vitroassay to qualitatively or quantitatively detect LPS in a sample.

[0029] Another object of the invention is to provideendotoxin-neutralizing proteins that can be used to produceendotoxin-free solutions and tools for use in, for example, variousmedical applications.

[0030] An advantage of the present invention is that theendotoxin-neutralizing proteins have a half-life in serum which isenhanced relative to the half-life of naturally-occurring LPS-bindingproteins, and bind LPS without triggering a significant, undesirableimmune response.

[0031] Another advantage of the invention is that the RENPs can beadministered to a patient immediately upon identification of a symptomof an endotoxin-associated disorder.

[0032] Another advantage is that the endotoxin-neutralizing proteins canbe administered prophylactically to a patient at risk of endotoxic shockor other LPS-mediated condition.

[0033] An advantage of the invention is that various RENPs havingbinding specificity for LPS for detection of LPS either in vivo or invitro.

[0034] Another advantage of the invention is that the RENPs can beattached to a variety of detectable labels.

[0035] Yet another advantage of the invention is that the RENPs can bebound to a molecule which can interact with or which can be a portion ofa solid support.

[0036] These and other objects, advantages and features of the presentinvention will become apparent to those persons skilled in the art uponreading the details of the vectors, cell lines and methodology as morefully set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0037] FIGS. 1A-1D are a comparison of the amino acid sequences of humanLBP as described by Schumann et al. (LBPa) and as used herein (LBPb).

[0038]FIG. 2 is a schematic diagram showing the various combinations ofBPI, LBP, BPI variants, and/or LBP variants which can be used togenerate an RENPs of the invention.

[0039] FIGS. 3A-3D show the nucleotide and amino acid sequences of BPI.

[0040] FIGS. 4A-4C show the nucleotide and amino acid sequences of LBP.

[0041] FIGS. 5A-5F are a comparison of the amino acid sequences of BPIand LBP from various species.

[0042]FIG. 6 shows the amino acid sequence of L₁₋₁₉₇B₂₀₀₋₄₅₆ (NCY118).

[0043]FIG. 7 is a graph showing the effects of BPI, LBP,L_(1-197(I43→V))B_(200-456(N206→D)) (NCY103) and B₁₋₁₉₉L₂₀₀₋₄₅₆ (NCY104)on ^(biotinylated)BPI binding to LPS.

[0044]FIG. 8 is graph showing the effects of BPI, LBP,L_(1-197(I43→V))B_(200-456(N206→D)) (NCY103), B₁₋₁₉₉L₂₀₀₋₄₅₆ (NCY104),or B_((S351→A)) (NCY105) protein on LPS activity in the chromogenic LALassay.

[0045]FIG. 9 is a graph showing FITC-LPS binding to monocytes in thepresence of BPI or L_(1-197(I43→V))B_(200-456(N206→D)) (NCY103).

[0046]FIG. 10 is a graph showing the effects of BPI, LBP,L_(1-197(I43→V))B_(200-456(N206→D)) (NCY103) or B₁₋₁₉₉L₂₀₀₋₄₅₆ (NCY104),on TNF release by LPS in whole blood.

[0047]FIG. 11 is a graph showing clearance of BPI, LBP,L_(1-197(I43→V))B_(200-456(N206→D)) (NCY103) or B₁₋₁₉₉L₂₀₀₋₄₅₆ (NCY104)from mouse serum after intravenous injection.

[0048]FIG. 12 is a graph comparing the efficacy of BPI andL_(1-197(I43→V))B_(200-456(N206→D)) (NCY103) in the protection toendotoxin challenge.

[0049] FIGS. 13A-13C are graphs showing the effects of BPI,L_(1-197(I43→V))B_(200-456(N206→D)) (NCY103), L₁₋₁₉₇B₂₀₀₋₄₅₆ (NCY118),L₁₋₁₉₈B₂₀₁₋₄₅₆Fc (NCY144), L₁₋₅₉B₆₀₋₄₅₆ (NCY114), L₁₋₁₃₄B₁₃₅₋₄₅₆(NCY115), L₁₋₃₅₉B₃₆₀₋₄₅₆ (NCY117), and B_(CAT9) (NCY139) on biotinylatedBPI binding to LPS.

[0050] FIGS. 14A-14B are graphs showing the effects of BPI, LBP,L_(1-197(I43→V))B_(200-456(N206→D)) (NCY103) and B₁₋₁₉₉L₂₀₀₋₄₅₆ (NCY104)on FITC-labeled LPS binding to human peripheral blood monocytes in thepresence of 10% autologous serum (14A) and in the absence of serum andpresence of 0.5% human serum albumin (14B).

[0051]FIG. 15 is a graph comparing the effects of LBP vs.L_(1-197(I43→V))B_(200-456(N206→D)) (NCY103), B₁₋₁₉₉L₂₀₀₋₄₅₆ (NCY104),L₁₋₃₅₉B₃₆₀₋₄₅₆ (NCY117) and PLL (poly-L-lysine) on the stimulation ofTNFα release by phorbol ester-induced THP-1 cells in the absence ofserum.

[0052]FIG. 16 is a graph showing the effects of variousrecombinant-endotoxin neutralizing proteins upon LPS-mediated TNFproduction in THP-1 cells cultured without serum.

[0053] FIGS. 17A-17H are graphs showing the clearance of: BPI, LBP,L_(1-197(I43→V))B_(200-456(N206→D)) (NCY103), B₁₋₁₉₉L₂₀₀₋₄₅₆ (NCY104),and L₁₋₁₉₇B₂₀₀₋₄₅₆ (NCY118) (17A); BPI, L₁₋₅₉B₆₀₋₄₅₆ (NCY114),L₁₋₁₃₄B₁₃₅₋₄₅₆ (NCY115), and B_(CAT9) (NCY139) (17B); BPI, LBP,L₁₋₃₅₉B₃₆₀₋₄₅₆ (NCY117) and L₁₋₁₉₇B₂₀₀₋₄₅₆ (NCY118) (17C); and BPI LBPand L₁₋₁₉₈B₂₀₁₋₄₅₆Fc (NCY144) (assayed for both Fc and BPI) in CD-1 mice(17D) ; LBP, L₁₋₂₇₅B₂₇₈₋₄₅₆ (NCY116), L₁₋₃₅₉B₃₆₀₋₄₅₆ (NCY117),L₁₋₁₉₇B₂₀₀₋₄₅₆ (NCY118) (17E); LBP, L_(1-197(I43→V))B_(200-456(N206→D))(NCY103), L₁₋₁₃₄B₁₃₅₋₄₅₆ (NCY115), L₍₁₋₁₉₈₎B(202-275)L₍₂₇₄₋₄₅₆₎(NCY135), and L₍₁₋₁₃₄₎B₍₁₃₆₋₂₇₅₎L₍₂₇₄₋₄₅₆₎ (NCY134) (17F); LBP (NCY102),L_(CAT6) (NCY141), L_(CAT9) (NCY142), L_(CAT15) (NCY143) and BPI (17G);and BPI, L₁₋₁₃₄B₁₃₅₋₄₅₆ (NCY115), and L₁₋₅₉B₆₀₋₄₅₆ (NCY114) (17H).

[0054]FIG. 18 is Western blot of BPI and L₁₋₁₉₇B₂₀₀₋₄₅₆ (NCY118)produced in Pichia pastoris.

[0055]FIG. 19 is a graph showing the effects of BPI andL_(1-197(I43→V))B_(200-456(N206→D)) (NCY103) on endotoxin activation ofmonocytes.

[0056]FIG. 20 is a graph showing the protective effects ofL₁₋₁₉₇B₂₀₀₋₄₅₆ (NCY118) to endotoxin challenge in mice.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Before the present recombinant endotoxin-neutralizing proteins,methods for providing therapy to a patient suffering from anendotoxin-related disorder, and compositions and method for diagnosis ofa condition associated with LPS are described, it is to be understoodthat this invention is not limited to the particular methodology,protocols, cell lines, vectors and reagents described as such may, ofcourse, vary. It is also to be understood that the terminology usedherein is for the purpose of describing particular embodiments only, andis not intended to limit the scope of the present invention which willbe limited only by the appended claims.

[0058] It must be noted that as used herein and in the appended claims,the singular forms “a”, “and”, and “the” include plural referents unlessthe context clearly dictates otherwise. Thus, for example, reference to“a recombinant endotoxin-neutralizing protein” includes a plurality ofsuch proteins and reference to “the DNA encoding the recombinantendotoxin-neutralizing protein” includes reference to one or moretransformation vectors and equivalents thereof known to those skilled inthe art, and so forth.

[0059] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this invention belongs. Although any methods,devices and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention, the preferredmethods, devices and materials are now described.

[0060] All publications mentioned herein are incorporated herein byreference for the purpose of describing and disclosing the cell lines,vectors, and methodologies which are described in the publications whichmight be used in connection with the presently described invention.

Definitions

[0061] By “lipopolysaccharide” or “LPS” is meant a compound composed ofa heteropolysaccharide (which contains somatic O antigen) covalentlybound to a phospholipid moiety (lipid A). LPS is a major component ofthe cell wall of Gram-negative bacteria.

[0062] By “endotoxin” is meant a heat-stable toxin associated with theouter membranes of certain Gram-negative bacteria, including theenterobacteria, brucellae, neisseriae, and vibrios. Endotoxin, normallyreleased upon disruption of the bacterial cells, is composed oflipopolysaccharide molecules (LPS) and any associated proteins. Thephospholipid moiety of LPS, lipid A, is associated with LPS toxicity.When injected in large quantities endotoxin produces hemorrhagic shockand severe diarrhea; smaller amounts cause fever, altered resistance tobacterial infection, leukopenia followed by leukocytosis, and numerousother biologic effects. Endotoxin is a type of “bacterial pyrogen,”which is any fever-raising bacterial product. The terms “endotoxin,”“LPS,” and “lipopolysaccharide” as used herein are essentiallysynonymous.

[0063] By “recombinant endotoxin-neutralizing polypeptide”, “RENP” or“recombinant LPS-neutralizing polypeptide” is meant a protein which hasbeen genetically engineered and contains an LPS-binding domain.Preferably, such recombinant LPS-binding proteins bind endotoxin, have arelatively long half-life in serum (e.g., compared tobactericidal/permeability increasing (BPI) protein), and elicit no orrelatively little of the undesirable inflammatory side effectsassociated with endotoxin and/or binding of LPS to particular naturallyoccurring endotoxin-binding proteins (e.g., lipopolysaccharide binding(LBP) protein). “RENPs” of the invention do not occur naturally and aredistinct from those endotoxin-binding proteins that do occur in nature,specifically BPI and LBP.

[0064] By “LPS-binding domain” is meant an amino acid sequence whichconfers specific and selective LPS binding upon a polypeptide.

[0065] By “high affinity LPS binding” is meant an LPS binding affinitygreater than the LPS binding affinity of LBP, preferably about the sameor greater than the LPS binding affinity of BPI.

[0066] By “endotoxin-neutralizing activity” is meant a biologicalactivity associated with inhibition of the toxic effects oflipopolysaccharide, e.g., by binding LPS and preventing interaction ofLPS with proteins and/or receptors which mediate an undesirableimmunological response associated with endotoxin in a mammalian host.

[0067] By “recombinant” or “genetically engineered” is meant a DNAsequence, or a polypeptide encoded thereby, generated using nucleic acidmanipulation techniques (e.g., cloning, PCR, and/or fusion proteintechniques). “Recombinant” or “genetically engineered” DNA, and thus theproteins encoded by such DNAs, do not occur in nature.

[0068] By “half-life” is meant the time required for a living tissue,organ, or organism to eliminate one-half of a substance introduced intoit.

[0069] By “molecule which enhances the half-life” or “half-lifeenhancing molecule” is meant chemical moiety (e.g., bound via a chemicalmodification) which enhances the biological half-life of a polypeptidewith which it is associated relative to the biological half-life of theparent polypeptide. Chemical moieties include an amino acid sequence orprotein. For example, where a polyethylene glycol (PEG) moiety iscovalently bound to a protein so as to increase the half-life of theprotein relative to the un-PEGylated parent protein, the PEG moiety isthe “molecule which enhances the half-life” of the protein.

[0070] By “half-life determining portion” of a polypeptide is meant anamino acid sequence which is associated with the biological half-life ofthe polypeptide.

[0071] By “bactericidal/permeability increasing protein” or “BPI” ismeant a naturally occurring or recombinantly expressed protein havingthe DNA and amino acid sequences shown in FIGS. 3A-3D.

[0072] By “lipopolysaccharide binding protein” or “LBP” is meant anaturally occurring or recombinantly expressed protein having the DNAand amino acid sequences shown in FIGS. 1A-1D and FIGS. 4A-4C.

[0073] By “BPI variant” is meant a protein having an amino acid sequencesimilar to, but not identical to, the amino acid sequence of BPI. “BPIvariants” (a) bind LPS, (b) competitively bind LPS in the presence ofBPI or LBP, and (c) inhibit the LPS-mediated production of TNFα by humanmonocytes. In general, “BPI variants” contain the amino acid sequence ofBPI but with at least one of: 1) an amino acid substitution; 2) an aminoacid deletion; or 3) an amino acid addition, relative to the BPI aminoacid sequence.

[0074] By “LBP variant” is meant a protein having an amino acid sequencesimilar to, but not identical to, the amino acid sequence of LBP. “LBPvariants” (a) bind LPS, (b) competitively bind LPS in the presence ofBPI or LBP, and (c) inhibits production of TNFα by human monocytes. Ingeneral, “LBP variants” contain the amino acid sequence of LBP but withat least one of: 1) an amino acid substitution; 2) an amino aciddeletion; or 3) an amino acid addition, relative to the LPB amino acidsequence.

[0075] By “detectable label” is meant any molecule recognized in the artas a means for identifying and/or detecting a protein to which thedetectable label is bound. Exemplary “detectable labels” includeradionucleotides, fluorescent moieties, biotin, and antigenic molecules(e.g., a polypeptide which is specifically bound by an anti-polypeptideantibody). “Detectable labels” include a portion of a chimeric proteinwhere a portion of the chimeric protein can be detected by, for example,binding of a detectably labeled antibody or other detectably labeledmolecule which specifically binds the chimeric protein portion.

[0076] By “support” is meant a surface to which LPS or an RENP of theinvention can be bound and immobilized. Exemplary supports includevarious biological polymers and non-biological polymers.

[0077] By “condition associated with endotoxin”, “endotoxin associateddisorder”, or “endotoxin-related disorder” is meant any conditionassociated with extra-gastrointestinal (e.g., mucosal, blood-borne,closed space) lipopolysaccharide, e.g., a condition associated withbacteremia or introduction of lipopolysaccharide into the blood streamor onto an extra-gastrointestinal mucosal surface (e.g., the lung). Suchdisorders include, but are not limited to, endotoxin-related shock,endotoxin-related disseminated intravascular coagulation,endotoxin-related anemia, endotoxin-related thrombocytopenia,endotoxin-related adult respiratory distress syndrome, endotoxin-relatedrenal failure, endotoxin-related liver disease or hepatitis, systemicimmune response syndrome (SIRS) resulting from Gram-negative infection,Gram-negative neonatal sepsis, Gram-negative meningitis, Gram-negativepneumonia, neutropenia and/or leucopenia resulting from Gram-negativeinfection, hemodynamic shock and endotoxin-related pyresis.

[0078] By “transformation” is meant a permanent genetic change inducedin a cell following incorporation of new DNA (i.e., DNA exogenous to thecell). Where the cell is a mammalian cell, the permanent genetic changeis generally achieved by introduction of the DNA into the genome of thecell.

[0079] By “transformed cell” is meant a cell into which (or into anancestor of which) has been introduced, by means of recombinant DNAtechniques, a DNA molecule encoding a protein of interest.

[0080] By “promoter” is meant a minimal DNA sequence sufficient todirect transcription. “Promoter” is also meant to encompass thosepromoter elements sufficient for promoter-dependent gene expressioncontrollable for cell-type specific, tissue-specific or inducible byexternal signals or agents; such elements may be located in the 5′ or 3′regions of the native gene.

[0081] By “operably linked” is meant that a DNA sequence and aregulatory sequence(s) are connected in such a way as to permit geneexpression when the appropriate molecules (e.g., transcriptionalactivator proteins) are bound to the regulatory sequence(s).

[0082] By “operatively inserted” is meant that the DNA of interestintroduced into the cell is positioned adjacent a DNA sequence whichdirects transcription and translation of the introduced DNA (i.e.,facilitates the production of, e.g., a polypeptide encoded by a DNA ofinterest).

[0083] By “mammalian subject” or “mammalian patient” is meant any mammalfor which the therapy of the invention is desired, including human,bovine, equine, canine, and feline subjects.

[0084] The invention will now be described in further detail.

[0085] Nomenclature used to describe RENPs

[0086] In order to facilitate the discussion and description of theRENPs of the invention, each RENP is designated a specific formula tobriefly describe the amino acid sequence of the protein, as well as theorigin of specific portions of the protein. The portion of BPI in therecombinant protein is designated with the letter B, followed by anamino acid sequence numbering assignment corresponding to that shown inFIGS. 5A-5F for human BPI, wherein the mature N-terminus is designatedas residue 1. The portion of LBP in certain LBP variants and chimeras isdesignated by the letter L, followed by an amino acid sequence numberingassignment corresponding to that shown in FIGS. 1A-1D for human LBP,wherein the mature N-terminus is designated as residue 1. To avoidconfusion between the erroneous LBP amino acid sequence published bySchumann et al., supra (designated LBP-a) and the correct LBP amino acidsequence used in the RENPs of the invention (designated LBP-b) andpresented in FIGS. 1A-1D. The differences between the DNA and amino acidsequences for “LBP-a” and “LBP-b” are presented in Table 2A below.

[0087] As an example of RENP nomenclature, L₁₋₁₉₇B₂₀₀₋₄₅₆ (NCY118)contains amino acid residues 1-199 of LBP fused at the C-terminus of theLBP portion to the N-terminus of amino acid residues 200-456 of BPI.L₁₋₁₉₇B₂₀₀₋₄₅₆, shown in FIG. 6 has the N-terminal domain of LBP (havinga putative endotoxin-binding domain) fused to the C-terminal domain ofBPI (having a putative LPS-clearing domain).

[0088] In this application, single amino acid residue substitutions arenoted in parentheses, wherein the original amino acid residue isindicated (using the standard one letter code for amino acids), followedby the substitute amino acid residue. For example, the BPI varianthaving an alanine residue substituted for the original serine residue atposition 351 (which substitution removes a glycosylation signal) isdesignated BPI_((S351→A)). In another example, in B_((DS200→DP)), aproline residue is substituted for the serine residue at position 200.In this latter example, the amino acid substitution produces a formicacid-cleavable site.

[0089] As another example, the RENP LBP-BPI chimera NCY103 is designatedL_(1-198(I43→V))B_(201-456(D206>N)). In the recombinant protein, theoriginal isoleucine residue at position 43 of the LBP portion issubstituted with a valine residue, and the original asparagine residueat position 206 of the BPI portion is substituted with an aspartateresidue. The C-terminus of the LBP amino acid sequence 1-198 havingisoleucine substituted at position 43 is covalently bound to theN-terminus of the BPI amino acid sequence 201-456 having valinesubstituted at position 206.

[0090] The amino acid substitutions may be substitutions wherein anoriginal amino acid residue at a given position is substituted with theresidue at the corresponding position in a different protein.BPI_((Xn→Y)) is an example of such a substitution, wherein amino acidresidue X at position n in BPI is substituted with residue Y which isfound at position n in LBP (or rabbit or bovine LBP). “X” and “Y” denoteamino acid positions in a primary amino acid sequence. “Y” as used inthis context is not to be confused with the symbol “Y” denoting theamino acid residue tyrosine. LBP_((Xn→Y)) is another example of such asubstitution, wherein amino acid residue X at position n in LBP issubstituted with residue Y which is found at position n in BPI (orrabbit or bovine BPI).

[0091] Amino acid residue insertion changes are noted in parentheses, byindicating the amino acid residue after which the insertion occurs,followed by the amino acid residue after which the insertion occurstogether with the inserted residue or residues. For example,B_((D200papain)) indicates that an amino acid sequence for cleavage ofthe BPI variant by papain is inserted after the aspartic acid at residueposition 200. TABLE 2A Individual Sequence Differences Between Schumannet al. and LBP as Used Herein NUCLEIC ACID PROTEIN Alpha Beta Alpha BetaA₄₂ C₄₂ G₁₂₉YCL₁₃₂ V₁₂₉TAS₁₃₂ C₃₁₈ T₃₁₈ S₁₄₉ F₁₄₉ G₄₈₈ (np) A₂₄₁V₂₄₁MSLP₂₄₅ (np) C₄₉₉ L₄₁₁ F₄₁₁ T₅₄₆ C₅₄₆ C₅₄₈ T₅₄₈ (np)T₈₂₄CATGAGCCTTC₈₃₅ C₁₃₃₃ T₁₃₃₃

[0092] Table 2B describes some exemplary general classes of RENPs of theinvention. In the formulas in Table 2B, n represents an amino acidresidue position in the mature sequence of BPI or LBP, x represents anamino acid residue in a position which is C-terminal to n in thesequence of BPI or LBP, and y represents an amino acid residue in aposition which is C-terminal to x in the sequence of BPI or LBP. Thesymbols n, x and y denote the amino acid residue positions as they occurin the mature sequence of the native protein, and not necessarily thepositions as they occur in the variant protein. TABLE 2B Examples ofRENPs+HZ,1/32 BPI variant (N-terminal frag.) B_(1 − n) LBP variant(N-terminal frag.) L_(1 − n) BPI variant (C-terminal frag.) B_(n − 456)LBP variant (C-terminal frag.) L_(n − 456) BPI variant (internal frag.)B_(n − x) LBP variant (internal frag.) L_(n − x) LBP-BPI chimeraL_(n − x)B_((x + 1)−y) BPI-LBP chimera B_(n − x)L_((x + 1)−y) LBP-BPIchimera L_(n − x)B_((x + 1)−456) BPI-LBP chimeraB_(n − x)L_((x + 1)−456) LBP-BPI chimera L_(1 − n)B_((n + 1)−x) BPI-LBPchimera B_(1 − n)L_((n + 1)−x) LBP-BPI chimera L_(1 − n)B_((n + 1)−456)BPI-LBP chimera B_(1 − n)L_((n + 1)−456) LBP-BPI-LBP chimeraL_(1 − n)B_((n + 1)−x)L_((x + 1)−456) BPI-LBP-BPI chimeraB_(1 − n)L_((n + 1)−x)B_((x + 1)−456)

[0093] All of the constructs in Table 2B can also contain additionalmolecules which confer an enhanced half-life upon the RENP (e.g., theRENP can be covalently bound to a polyethylene glycol moiety, or aportion of an immunoglobulin protein or other amino acid sequence whichconfers a half-life increased relative to the unmodified protein). Thegeneral scheme for generation of RENPs is outlined in FIG. 2.

[0094] Production of RENPs

[0095] The RENPs of the invention minimally have characteristicsassociated with (i) specific and high affinity binding tolipopolysaccharide and (ii) endotoxin-neutralizing activity. In general,the amino acid sequence of RENPs is based upon an amino acid sequence ofBPI, LBP, or both. However, the amino acid sequences of the RENPs aredistinct from that of BPI and LBP, i.e. the RENPs contain amino acidsubstitutions, deletions, and/or additions relative to the amino acidsequence of BPI or LBP. Thus, the RENPs of the invention contain: 1)amino acid sequences of a naturally-occurring LPS-binding protein (i.e.,LBP and/or BPI); and/or 2) amino acid sequences which do not occurwithin a single naturally-occurring LPS-binding protein (i.e., LBP orBPI). RENPs can thus be similar to, but not identical to, LBP or BPI.For example, the RENPs can be fragments of BPI and/or LBP, as the aminoacid sequences of such RENPs are similar to, but not identical to,naturally occuring BPI or LBP. Moreover, the RENPs of the inventiongenerally have biological properties distinct from and advantageous toeither BPI or LBP. RENPs of the invention include BPI variants, LBPvariants, and chimeric proteins composed of amino acid sequences derivedfrom BPI, LBP, BPI variants, and/or LBP variants.

[0096] For example, RENPs can contain an amino acid sequence of BPI,where the BPI amino acid sequence 1) has been altered at a site ofglycosylation (e.g., insertion or deletion of a glycosylation site); 2)contains a neutral or anionic amino acid substituted at a cationicresidue of the BPI amino acid sequence (cationic substitution variants);3) contains an amino acid substitution at a position normally occupiedby cysteine in the BPI sequence (cysteine substitution variants); 4)contains an amino acid substitution where the substituted amino acid isthe amino acid at the corresponding position in the LBP amino acidsequence; and/or 5) contains an insertion or deletion of one or moresecondary structure-altering amino acid residues.

[0097] Exemplary BPI variants containing a glycosylation site alterationinclude BPI variants having an amino acid residue other than serinesubstituted for the serine residue at position 351 of the BPI amino acidsequence. BPI variants of this type are of the formula BPI(S351→X),wherein X is any amino acid other than serine. Preferably, the aminoacid substituted at position 351 is alanine. Other BPI variants having aglycosylation site deleted can be generated by, for example, other aminoacid substitutions within the glycosylation site.

[0098] Additional exemplary BPI variants contain a neutral or anionicamino acid substituted at a cationic residue of the BPI amino acidsequence (cationic substitution variants). For example, one or more ofthe nonconserved positively-charged residues in BPI (i.e., thoseresidues not found at the corresponding positions in LBP) can besubstituted with the corresponding residue or residues in LBP, thusrendering BPI less cationic. Preferably, the cationic substitutionvariant contains an amino acid substitution in at least one of BPI aminoacid residue positions 27, 30, 33, 42, 44, 48, 59, 77, 86, 90, 96, 118,127, 148, 150, 160, 161, 167, 169, 177, 185, or 198. The cationicsubstitution variant can contain multiple amino acid substitutions. Forexample, the cationic substitution variant can contain a neutral oranionic residues at 1) BPI amino acid residue positions 27, 30, 33, 42,44, 48, and 59; 2) BPI amino acid residue positions 77, 86, 90, 96, 118,and 127; 3) BPI amino acid residue positions 148, 150, 160, 161, 167,169, 177, 185, and 198; or 4) BPI amino acid residue positions 27, 30,33, 42, 44, 48, 59, 77, 86, 90, 96, 118, 127, 148, 150, 160, 161, 167,169, 177, 185, and 198.

[0099] Further example BPI variants contain an amino acid substitutionat a position normally occupied by cysteine in the BPI sequence(cysteine mutant). The amino acid selected for substitution at this sitecan be the amino acid in the corresponding position in LBP. For example,a cysteine residue in BPI (which is not conserved in LBP) may besubstituted with an alanine residue (the corresponding residue in LBP).Preferably, the amino acid substitution is at a cysteine residue at BPIamino acid residue position 132, 135, or 175. Preferably, alanine orserine is substituted for cysteine. More preferably, alanine issubstituted for the cysteine at position 132 of BPI. Cysteinesubstitution mutants of BPI can prevent aggregation of the resultingRENPs during their production or use.

[0100] Another example of a BPI variant includes a BPI variant having anamino acid substitution where the substituted amino acid is the aminoacid at the corresponding position in LBP. The amino acid at thecorresponding position is determined by aligning the BPI and LBP aminoacid sequences so as to maintain the highest level of amino acidsequence identity between the two sequences. For example, an RENP havingthe formula B_((Q329→S)) contains a substitution of the glutamine at BPIresidue position 329 with the serine residue at the corresponding LBPresidue position 327 (see FIGS. 5A-5F).

[0101] Additional exemplary BPI variants contain an insertion ordeletion of one or more secondary structure-altering amino acidresidues. For example, one or more of the nonconserved proline residuesin BPI may be substituted with the corresponding non-proline residue inLBP.

[0102] Alternatively, or in addition to the amino acid sequence of BPIand/or a BPI variant, the RENPs can contain an amino acid sequence ofLBP, where the LBP amino acid sequence 1) has been altered at a site ofglycosylation (e.g., insertion or deletion of a glycosylation site); 2)contains a cationic amino acid substituted at a neutral or anionic aminoacid of the LBP amino acid sequence (cationic replacement mutant); 3)contains an amino acid substitution where the substituted amino acid isthe amino acid at the corresponding position in the BPI amino acidsequence; and/or 4) contains an insertion or deletion of one or moresecondary structure-altering amino acid residues. The LBP DNA and aminoacid sequence used in the construction of particular RENPs exemplifiedherein is the amino acid sequence of human LBP in FIGS. 5A-B.

[0103] Exemplary LBP variants contain a cationic amino acid substitutedat a neutral or anionic amino acid of the LBP amino acid sequence(cationic replacement variant). For example, one or more of thenonconserved amino acid residues in LBP (at a position which correspondsto a positively-charged residue in BPI) may be substituted with thecorresponding positively-charged residue in BPI, and thus result in anLBP variant having an increased positive charge, thus enhancing bindingto the negatively charged phosphate groups in LPS, and/or facilitatinginteraction with the negatively charged surfaces of Gram-negativebacteria. Positively-charged residues include, by way of example,lysine, arginine, and histidine. Preferably, the substituted cationicamino acid is at least one of LBP amino acid residue positions 77, 86,96, 118, 126, 147, 148, 158, 159, 161, 165, 167, 175, 183, or 196.Cationic replacement variants can contain multiple amino acid residuesubstitutions at any combination of the amino acid residues recitedabove.

[0104] Other exemplary LBP variants include an LBP variant having anamino acid substitution where the substituted amino acid is the aminoacid at the corresponding position in BPI. For example, L_((A401→D))contains a substitution of the alanine residue of LBP at position 401with the aspartic acid residue at the corresponding BPI residue position403.

[0105] Further exemplary LBP variants contain an insertion or deletionof one or more one or more secondary structure-altering amino acidresidues. For example, one or more of the nonconserved amino acidresidues in LBP (at a position which corresponds to a proline in BPI)may be substituted with a proline residue. Preferably, such amino acidalterations alter the secondary structure of the resulting LBP variantso that it is more like the secondary structure of BPI.

[0106] Preferably, the RENPs of the invention contain at least oneLPS-binding domain of BPI, LBP, a BPI variant, and/or a LBP variant. Forexample, the LPS-binding domain can be derived from BPI and/or LBP aminoacid sequences 17-45, 65-99, and/or 141-167. Preferably, the RENP has anLPS binding affinity that is greater than the LPS binding affinity ofLBP, more preferably an LPS binding affinity that is the same or greaterthan the LPS binding affinity of BPI. Preferably, the RENP has an LPSbinding affinity that is about 25-fold to 50-fold, preferably about50-fold to 100-fold, more preferably about 100-fold to 300-fold greaterthan the LPS binding affinity of LBP as determined by LPS binding or LPSbinding competition assays. The LPS binding affinity of BPI is about60-fold to 100-fold greater than the LPS binding affinity of LBP.

[0107] The RENPs can contain multiple LPS-binding domains derived fromany of these LPS-binding proteins. For example, an RENP can be amultivalent chimeric protein (i.e., a fusion protein) composed of anLPS-binding domain of BPI covalently bound (i.e., as in a fusionprotein) to an LPS-binding domain of LBP. As used herein, a chimerameans a protein comprising all or a portion of a first protein fused toall or a portion of a second protein, which resulting fusion protein mayin turn be fused to all or a portion of a third protein. Examples ofchimeras include, by way of example, (a) a protein comprising a portionof LBP fused to a portion of BPI, (b) a protein comprising a portion ofLBP fused to a portion of BPI which portion of BPI is in turn fused to aportion of an immunoglobulin protein, or (c) a protein comprising aportion of LBP fused to a portion of BPI, which is in turn fused to aportion of LBP. Each protein portion of the chimera may comprise afragment of the protein, a point mutant of the protein (i.e., avariant), a deletion mutant of the protein, or a point and deletionmutant of the protein.

[0108] Examples of BPI fragments which can be incorporated into theRENPs of the invention include the BPI amino acid sequences 1-25, 1-85,1-137, 1-135, 1-147, 1-1-59, 88-100, 148-161, 137-199, 44-1-59, 44-199,135-199, 100-199, 162-199, 100-147. Examples of LBP fragments which canbe incorporated into the RENPs of the invention include LBP amino acidsequences 1-43, 1-87, 26-135, 26-134, 86-99, 101-146, 101-197, 135-197,137-197, 158-197, 160-197, and/or 147-159. The amino acid sequences ofBPI and/or LBP can be comined in any order from N- to C-terminus toprovide an RENP having sequences derived from BPI and/or LBP. Forexample, the RENPs can have the sequences B₁₋₁₃₇L₁₃₇₋₁₉₇, L₁₋₄₃B₄₄₋₁₉₉,B₁₋₁₅₉L₁₅₈₋₁₉₇, B₁₋₁₃₅L₁₃₅₋₁₉₇, L₁₋₄₃B₄₄₋₁₅₉L₁₅₈₋₁₉₇,B₁₋₂₅L₂₆₋₁₃₅B₁₃₇₋₁₉₉, B₁₋₂₅L₂₆₋₁₃₄B₁₃₅₋₁₉₉,L₁₋₈₇B₈₈₋₁₀₀L₁₀₁₋₁₄₆B₁₄₈₋₁₆₁L₁₆₀₋₁₉₇, B₁₋₈₅L₈₆₋₉₉B₁₀₀₋₁₉₉,B₁₋₁₄₇L₁₄₇₋₁₅₉B₁₆₂₋₁₉₉, B₁₋₈₅L₈₆₋₉₉B₁₀₀₋₁₄₇L₁₄₇₋₁₅₉B₁₆₂₋₁₉₉,L₁₋₈₇B₈₈₋₁₀₀L₁₀₁₋₁₉₇, or various combinations of other BPI and/or LBPfragments.

[0109] RENPs can share properties of both BPI and LBP. For example,fusing the N-terminal domain of LBP to the C-terminal domain of BPIresults in an RENP which differs from LBP in that the chimeraneutralizes endotoxin in whole blood and differs from BPI in that thechimera has a longer circulating half-life in vivo. Such RENPs havesignificant diagnostic and therapeutic potential. As per thenomenclature described above, RENPs designated BPI-LBP contain all or apart of the N-terminal domain of BPI fused to all or a part of theC-terminal domain of LBP. Likewise, RENPS designated LBP-BPI contain allor a part of the N-terminal domain of LBP fused to all or a part of theC-terminal domain of BPI.

[0110] Where the RENP contains amino acid sequences derived from bothBPI and LBP, the RENP is preferably composed of a C-terminal fragment ofBPI (or a BPI variant) and an N-terminal fragment of LBP (or an LBPvariant). Preferably the C-terminal fragment of BPI (or a BPI variant)contains amino acid residues 60-456, 136-456, 199-456, 277-456, 300-456,200-456, 136-361, 136-275, 200-275, or 200-361, more preferably 60-456,more preferably 199-359. The amino acid sequence of BPI from residue 199to residue 359 contains a region required for neutralizing LPS, i.e.,preventing LPS from stimulating an inflammatory response. Preferably,the N-terminal fragment of LBP (or an LBP variant) contains amino acidresidues 1-59, 1-134, 1-164, 1-175, 1-274, 1-359, 1-134, or 1-197, morepreferably 1-175. In addition to the specific amino acid sequences ofBPI and LBP recited above, the RENP can also contain amino acid residuesderived from the C-terminus of LBP (or an LBP variant), preferably LBP(or LBP variant) amino acid residues 360-456 or 274-456.

[0111] Polypeptides which bind LPS can be identified using any ofseveral assays well known in the art such as the 1) chromogenic LALcompetition assay, 2) binding to LPS immobilized on a surface, and 3)FITC-LPS assay for binding to macrophages. The ability of a polypeptideto neutralize endotoxin can also be determined using methods well knownin the art. Endotoxin neutralization assays include assays to examinethe ability of a polypeptide to 1) prevent LPS-induced TNF release inwhole blood, 2) inhibit or prevent TNF production by THP-1 cells, 3)provide protection in a mouse endotoxin challenge assay, and 4) reduceor prevent LPS-induced cytokine release and/or mortality in an animalmodel. Each of these assays are described in detail in the examplessection below. The results of the in vitro and in vivo assays usedherein are accepted in the art. The results of these assays arepredictive of relevant biological activity in vivo, e.g. in humans.

[0112] Preferably, the RENPs of the invention have a biologicalhalf-life (e.g., serum half-life) which is enhanced relative to thebiological half-life of BPI. Preferabl, the half-life of the RENP isenhanced relative to BPI such that the clearance time of the RENP is atleast 1.5-fold to 10-fold, preferably about 10-fold to 50-fold, morepreferably about 50-fold to 100-fold, even more preferably about100-fold to 350-fold slower than the clearance rate of BPI. Theclearance rate values representing these ranges are from about 8 ml/minto 1.5 ml/min, preferably 1.5 ml/min to 0.26 ml/min, more preferably0.26 ml/min to 0.13 ml/min, even more preferably about 0.13 ml/min to0.03 ml/min.

[0113] To enhance the RENP half-life, the RENP can be covalently boundto a molecule which enhances the half-life of the polypeptide. Thehalf-life enhancing molecule can be any of a variety of half-lifeenhancing molecules. Exemplary half-life enhancing molecules includeimmunoglobulin fragments, a half-life determining portion of LBP, ahalf-life determining portion of an LBP variant, or polyethylene glycol(PEG), preferably a half-life determining portion of LBP or an LBPvariant. Preferably, where the half-life enhancing molecule is a portionof LBP or an LBP variant, the half-life enhancing molecule is derivedfrom the N-terminus of the LBP or LBP variant amino acid sequence, morepreferably from amino acid residues 1-59, 1-134, 1-274, 1-359, 1-134,1-164, 1-175, or 1-197, most preferably 1-164 or 1-175. Methods ofattachment of PEG moieties to a protein (i.e., PEGylation) are wellknown in the art and are exemplified in U.S. Pat. Nos. 4,179,337;5,166,322; 5,206,344; and PCT application serial no. PCT/US94/11624,published Apr. 28, 1995.

[0114] As used herein, an RENP-Ig chimeric protein is an RENP which (i)contains a portion of BPI or LBP (at least 10 amino acid residues inlength of (a) BPI, or (b) BPI variant, or (c) LBP, and/or (d) LBPvariant) fused at the C-terminus to the N-terminus the Fc portion of animmunoglobulin molecule, and (ii) is capable of (a) binding to LPS, (b)competing with BPI or LBP for binding to LPS, and (c) inhibiting theproduction of TNFα by human monocytes. For example, the portion of theimmunoglobulin molecule is derived from an IgG molecule, specificallyfrom an IgG₁ heavy chain Fc domain. RENP-Ig chimera is a fusion proteincomposed predominantly of sequences derived from BPI, variant BPI, LBPand/or variant LBP. The term “LBP-BPI-IgG chimera” indicates that theRENP-Ig chimera contains amino acid sequences derived from both BPI (ora BPI variant) and LBP (or an LBP variant).

[0115] Identification of a half-life enhancing polypeptide sequence(e.g., a polypeptide derived from an immunoglobulin, LBP, or LBPvariant) can be accomplished using methods well known in the art. Forexample, the test polypeptide with and without the half-life enhancingmolecule bound to it are injected into an animal model to determine theeffects of the putative half-life enhancing molecule. If the half-lifeof the polypeptide with the molecule is enhanced relative to thehalf-life of the polypeptide without the molecule, then the molecule isa half-life enhancing molecule suitable for use in the RENPs of theinvention. For example, a putative half-life enhancing amino acidsequence is incorporated into a fusion protein with BPI. Both native BPIand the BPI fusion protein are injected into mice. If the BPI fusionprotein has a half-life significantly greater than the half-life ofnative BPI, then the amino acid sequence in the BPI fusion has half-lifeenhancing characteristics, and thus can be incorporated into the RENPsof the invention.

[0116] Vectors and constructs

[0117] Any nucleic acid vector can be used to express DNA encoding anRENP of the invention. The vectors containing the DNA sequence (or thecorresponding RNA sequence) which may be used in accordance with theinvention may be any prokaryotic or eukaryotic expression vectorcontaining the DNA (e.g., cDNA) or the RNA sequence of interest. Avariety of suitable vectors are publicly available and well known in theart. For example, a plasmid can be cleaved to provide linear DNA havingligatable termini. These termini are bound to exogenous DNA havingcomplementary, like ligatable termini to provide a biologicallyfunctional recombinant DNA molecule having an intact replicon and adesired phenotypic property.

[0118] A variety of techniques are available for DNA recombination inwhich adjoining ends of separate DNA fragments are tailored tofacilitate ligation. The vector is constructed using known techniques toobtain a transformed cell capable of expression of the RENP. Thetransformed cell is obtained by contacting a target cell with a RNA- orDNA-containing formulation permitting transfer and uptake of the RNA orDNA into the target cell. Such formulations include, for example,plasmids, viruses, liposomal formulations, or plasmids complexed withpolycationic substances such as poly-L-lysine or DEAC-dextran, andtargeting ligands. Transformed cells containing a construct encoding anRENP of the invention are also known in the art as “host vectorsystems”. Vectors for use in the construction of constructs encoding theRENPs of the invention, as well as methods for molecular cloning,nucleic acid manipulation, and transformation of both eukaryotic andprokaryotic host cells are well known in the art (see, for example,Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd Ed., 1989,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; herebyincorporated by reference with respect to bacterial and eukaryoticvectors, and methods and compositions for molecular cloning, nucleicacid manipulation, and transformation techniques).

[0119] The constructs of the invention may include promoter sequences toenhance expression of the RENP-encoding DNA, as well as other sequences(e.g., enhancers) which facilitate or enhance DNA expression. Inaddition, the RENP-encoding constructs can contain other components suchas a marker (e.g., an antibiotic resistance gene (such as an ampicillinresistance gene) or β-galactosidase) to aid in selection of cellscontaining and/or expressing the construct, an origin of replication forstable replication of the construct in a bacterial cell (preferably, ahigh copy number origin of replication), a nuclear localization signal,or other elements which facilitate production of the DNA construct, theprotein encoded thereby, or both.

[0120] In general, the RENPs of the invention are constructed from a DNAsequence encoding BPI, a BPI variant, LBP, an LBP variant, as well asvarious half-life enhancing molecules known in the art such asimmunoglobulin fragments. Both BPI and LBP have been cloned and theirDNA and amino acid sequences determined (FIGS. 3A-3B and 4A-4B,respectively). The DNA and amino acid sequences of numerousimmunoglobulins are known in the art. For example, the DNA sequence ofIgG, IgG_(2a), and IgG₄ are suitable for use to enhance the half-life ofthe RENPs of the invention.

[0121] Expression of recombinant endotoxin-neutralizing polypeptides

[0122] Techniques for obtaining expression of exogenous DNA or RNAsequences in a host cell are known in the art (see, for example,Sambrook et al., supra; hereby incorporated by reference with respect tomethods and compositions for eukaryotic and prokaryotic expression of aDNA encoding an RENP). Where the transformed cell is a prokaryotic hostcell, the preferred host is Escherichia coli. Where the transformed cellis a eukaryotic host cell, preferably the host cell is a mammalian cellor a yeast cell. Preferably, the mammalian host cell is a ChineseHamster Ovary (CHO) cell. Preferably, the yeast host cell is of thegenus Pichia, more preferably a strain of Pichia pastoris.

[0123] For prokaryotic expression, the construct should contain at aminimum a bacterial origin of replication and a bacterial promoteroperably linked to the RENP-encoding DNA. For eukaryotic expression, theconstruct should contain at a minimum a eukaryotic promoter operablylinked to a DNA of interest, which is in turn operably linked to apolyadenylation sequence. The polyadenylation signal sequence may beselected from any of a variety of polyadenylation signal sequences knownin the art. Preferably, the polyadenylation signal sequence is the SV40early polyadenylation signal sequence. The eukaryotic construct may alsoinclude one or more introns, which can increase levels of expression ofthe DNA of interest, particularly where the DNA of interest is a cDNA(e.g., contains no introns of the naturally-occurring sequence). Any ofa variety of introns known in the art may be used. Preferably, theintron is the human β-globin intron and inserted in the construct at aposition 5′ to the DNA of interest.

[0124] Purification of RENPs

[0125] Purification of the RENPs of the invention can be performedaccording to any of a variety of protein purification techniques knownin the art including gel electrophoresis, immunoprecipitation, ionexchange chromatography, affinity chromatography, or combinationsthereof (see, for example, Guide to Protein Purification, Deutscher,ed., Academic Press, Inc., San Diego, Calif., 1990). Preferably,purification of RENPs is accomplished by a combination of columnchromatographic techniques. For example, RENPs can be purified using afour-step purification procedure using 1) a cation exchange column(e.g., CM Sepharose), 2) an anion exchange column (e.g., Fast QSepharose), 3) a second cation exchange column (e.g., CM Sepharose), and4) a gel filtration sizing column (e.g., Sepharose CL₆B).

[0126] Pharmaceutical compositions

[0127] The RENPs of the invention can be formulated as an activeingredient in a pharmaceutical composition. In general, thepharmaceutical composition contains a therapeutically effective amountof an RENP and a pharmaceutically acceptable carrier. The pharmaceuticalcomposition can contain one or more RENPS. The amount of RENP whichconstitutes a therapeutically effective amount will vary according tothe time of administration (e.g., therapeutic or prophylacticadministration), the disease or condition to be treated, the route ofadministration, and various patient-dependent factors such as age,weight, gender, and severity of disease. Specific therapeuticallyeffective amounts appropriate for administration are readily determinedby one of ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th ed., Gennaro, ed., Mack PublishingCompany, Easton, Pa., 1990).

[0128] Pharmaceutically acceptable carriers suitable for use in theRENP-containing pharmaceutical compositions of the invention are wellknown to those skilled in the art. Selection of the pharmaceuticallyacceptable carrier will depend upon a variety of factors including theRENP to be administered, the route of administration, and the conditionto be treated.

[0129] Pharmaceutically acceptable carriers suitable for use with theRENPs of the invention include, but are not limited to, 0.01-0.1 M andpreferably 0.05 M succinate buffer or 0.8% saline. Additionally, suchpharmaceutically acceptable carriers may be aqueous or non-aqueoussolutions, suspensions, and emulsions. Further, pharmaceuticallyacceptable carriers may include detergents, phospholipids, fatty acids,or other lipid carriers. Examples of non-aqueous solvents are propyleneglycol, polyethylene glycol, vegetable oils such as olive oil, andinjectable organic esters such as ethyl oleate. Aqueous carriers includewater, alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils.

[0130] Pharmaceutically acceptable carriers for use with the RENPs ofthe invention include lipid carriers. A lipid carrier is anylipid-soluble substance which inhibits protein precipitation and inwhich the proteins of the subject invention are soluble. Lipid carrierscan be in the form of sterile solutions or gels, or can be detergents ordetergent-containing biological surfactants. Examples of nonionicdetergents include polysorbate 80 (also known as TWEEN 80 orpolyoxyethylenesorbitan monooleate). Examples of ionic detergentsinclude, but are not limited to, alykltrimethylammonium bromide.Exemplary lipid carriers and methods for solubilizing BPI, and thuswhich can be used in pharmaceutical compositions containing an RENP ofthe invention, are described in U.S. Pat. No. 5,234,912, incorporatedherein by reference.

[0131] Where the pharmaceutically acceptable carrier is a lipid carrier,the lipid carrier may be a liposome. A liposome is any phospholipidmembrane-bound vesicle capable of containing a desired substance, suchas BPI or BPI variant, in its hydrophilic interior. Intravenous vehiclesinclude fluid and nutrient replenishers, electrolyte replenishers suchas those based on Ringer's dextrose, and the like. Preservatives, otherpharmaceutically active compounds, and other additives may also bepresent, such as, for example, antimicrobials, antioxidants, chelatingagents, inert gases and the like.

[0132] Disease conditions amenable to treatment with RENPs

[0133] Various disease conditions are amenable to treatment using therecombinant LPS-neutralizing proteins of the invention. In general, anycondition of a mammalian subject (e.g., human, canine, feline, orbovine, preferably a human) which is associated with a toxic effect ofendotoxin can be treated by administration of the RENPs of theinvention. Endotoxin-related disorders amenable to treatment include,but are not limited to, endotoxin-related shock, endotoxin-relateddisseminated intravascular coagulation, endotoxin-related anemia,endotoxin-related thrombocytopenia, endotoxin-related adult respiratorydistress syndrome, endotoxin-related renal failure, endotoxin-relatedliver disease or hepatitis, systemic immune response syndrome (SIRS)resulting from Gram-negative infection, Gram-negative neonatal sepsis,Gram-negative meningitis, Gram-negative pneumonia, neutropenia and/orleucopenia resulting from Gram-negative infection, hemodynamic shock andendotoxin-related pyresis. Endotoxin-related pyresis is associated withcertain medical procedures, such as, for example, trans-urethralresection of the prostate, and gingival surgery. The presence ofendotoxin may result from infection at any site with a Gram-negativeorganism, or conditions which may cause ischemia of the gastrointestinaltract, such as hemorrhage, or surgical procedures requiringextracorporal circulation. The important role of endotoxin in hemorrhage(with endogenous LPS translocation from the gut), trauma, and sepsis iswell known. One skilled in the art can recognize additional conditionswhich can be treated using the therapy of the invention.

[0134] The recombinant, endotoxin-neutralizing proteins of the inventioncan also be administered to a patient prophylactically, e.g. to apatient at risk of an endotoxin-related disorder. For example, the RENPscan be administered to a patient who has a Gram-negative infection andis at risk of bacteremia, but who has not yet exhibited symptomsassociated with the toxic effects of endotoxin. The RENPs can also beadministered prior to surgery where the risk of introduction ofendotoxin into the patient is substantial. One of ordinary skill in theart can readily recognize other instances in which prophylacticadministration of a RENP is appropriate. The conditions which identifyan individual as being at risk of an endotoxin-related disorder are wellknown in the art.

[0135] Administration of RENPs

[0136] The recombinant, LPS-binding protein of the invention may beadministered using various methods well known in the art. U.S. Pat. Nos.5,171,739; 5,308,834; and 5,334,584; each incorporated herein byreference, describe methods and compositions for administration of BPI,and thus can provide additional guidance for administration of the RENPsof the invention. For example, the recombinant, LPS-binding protein canbe administered by injection or inhalation. Administration by injectioncan be an intravenous, intramuscular, or subcutaneous route, or bydirect injection directly into a site of infection (e.g., tissue or bodycavity). Preferably, injection is intravenous. Administration byinhalation is accomplished by delivery of the RENP to the lungs via anaerosol delivery system or via direct instillation. The aerosol may benebulized. Various devices and methods for aerosol drug delivery arewell known in the art. Methods for determining the appropriate route ofadministration and dosage are generally determined on a case-by-casebasis by the attending physician. Such determinations are routine to oneof ordinary skill in the art (see, for example, Remington'sPharmaceutical Sciences, 18th ed., Gennaro, ed., Mack PublishingCompany, Easton, Pa., 1990).

[0137] Therapeutically effective amounts of an RENP can be determinedaccording to methods well known to those skilled in the art. Specificdosages will vary according to a variety of factors, including the timeof administration (e.g., therapeutic or prophylactic administration),the disease or condition to be treated, the route of administration, theRENP to be administered, and various patient-dependent factors such asage, weight, gender, and severity of disease. The specific dosageappropriate for administration is readily determined by one of ordinaryskill in the art according to the factors discussed above (see, forexample, Remington's Pharmaceutical Sciences, 18th ed., Gennaro, ed.,Mack Publishing Company, Easton, Pa., 1990). In addition, the estimatesfor appropriate dosages in humans may be extrapolated fromdeterminations of the in vitro LPS binding affinity of the RENP used,the amount of the RENP effective to inhibit cytokine production bymononuclear cells in vitro, the amount of RENP effective to provideprotection to LPS challenge, and/or various other in vitro and in vivoassays indicative of the biological activity of the RENP.

[0138] In general, the amount of RENP administered is an amounteffective to bind LPS and thereby inhibit the undesirable biologicalactivities associated with LPS including monocyte and neutrophilactivation, TNF production, cytokine production, and other biologicalphenomena triggered by LPS in endotoxin-related disorders. Preferably,the amount of RENP administered is an amount effective to bind LBP andinhibit LPS-mediated stimulation of neutrophils and mononuclear cells.

[0139] In therapeutic administration of the RENPs of the invention, aneffective amount of an RENP is an amount effective to bind to LPS andthereby inhibit LPS-mediated stimulation of neutrophils and mononuclearcells in a subject having an endotoxin-related disorder. As used herein,“inhibit” means to inhibit at a level which is statistically significantand dose dependent. The terms “statistically significant” and “dosedependent” are well known to those skilled in the art. In general, aneffective amount of an RENP in a pharmaceutical composition fortreatment of a patient having an endotoxin-related disorder is an amountsufficient to deliver to the subject a recombinant protein of thesubject invention at a concentration of between about 0.1 mg/kg of bodyweight and about 100 mg/kg of body weight, preferably between about 1mg/kg of body weight and about 10 mg/kg of body weight. Preferably, theRENP(s) is administered by injection, infusion, or as an injected bolusso as to maintain a circulating RENP concentration of about 1-10 μg/ml.The preferred circulating RENP concentration can vary according to avariety of factors, including the LPS binding affinity of the specificRENP(s) administered.

[0140] As used herein, a prophylactically effective amount of an RENP ina pharmaceutical composition for the prevention of an endotoxin-relateddisorder is an amount effective to bind LPS and prevent LPS-mediatedbiological activity, e.g., LPS-mediated stimulation of monocytes andneutrophils. In general, a prophylactically effective amount of an RENPis an amount of a composition effective to deliver between about 0.1mg/kg of body weight and about 100 mg/kg of body weight, preferablybetween about 1 mg/kg of body weight and about 10 mg/kg of body weight,to the patient at risk of an endotoxin-related disorder.

[0141] The invention also provides an article of manufacture comprisingpackaging material and a pharmaceutical composition contained within thepackaging material. The packaging material includes a label whichindicates that the pharmaceutical composition can be used for treating asubject suffering from an endotoxin-related disorder and\or forpreventing an endotoxin-related disorder (e.g., inflammation) in asubject. The pharmaceutical composition contains a therapeuticallyeffective and/or prophylactically effective amount of an RENP and apharmaceutically acceptable carrier.

[0142] Assessment of therapy

[0143] The efficacy of the therapeutic or prophylactic use of the RENPsof the invention can be determined by monitoring patient symptomsassociated with an endotoxin-related disorder. Such symptoms, andmethods for monitoring, are well known in the art. For example, wherethe RENP is used in the treatment of a patient having anendotoxin-related disorder, the effectiveness of the RENP therapy can beassessed by monitoring fever, blood pressure, cytokine levels, and/orLPS levels in the patient's blood stream. The presence of LPS in theblood stream can be assayed as described above. Where the patient is notresponding, it may be desirable to increase the dosage of the RENPpharmaceutical composition or, where the patient is not respondingfavorably, discontinue the RENP regimen.

[0144] Detectably-labeled RENPs

[0145] Various detectable labels, as well as methods of attachment ofsuch labels to a protein, are well known in the art. Detectable labelscan be any molecule recognized in the art as a means for identifyingand/or detecting a protein to which the detectable label is bound.Exemplary “detectable labels” include, but are not limited toradionucleotides, fluorescent moieties, biotin, and antigenic molecules(e.g., a polypeptide which can be specifically bound by ananti-polypeptide antibody). Thus, detectable labels include a portion ofa chimeric protein (e.g., a fusion protein or genetically engineeredprotein) where a portion of the chimeric protein can be detected by, forexample, binding of a detectably labeled antibody or other detectablylabeled molecule which specifically binds the chimeric protein portion.For example, where the RENP contains a portion of the amino acidsequence of BPI, and an antibody which specifically binds that aminoacid sequence of BPI in the context of the RENP is available, the BPIamino acid sequence is the detectable label.

[0146] Methods for attaching (e.g., covalently binding) a detectablelabel to a protein are well known in the art. For example, methods forpreparation of ¹²⁵I-labeled proteins, biotin-labeled proteins, andFITC-labeled proteins are well known. Methods for detectably labelingantibodies are also well known in the art. Methods for the production ofantibodies for use in the subject invention (e.g., anti-BPI, anti-LBP,anti-BPI variant, anti-LBP variant, and anti-immunoglobulin fragmentantibodies) are well known in the art (see, for example, Antibodies: ALaboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y., 1988).

[0147] Detection of LPS in vitro

[0148] The detectably labeled RENPs of the invention can be used invarious methods for the detection of LPS either in vitro or in vivo.Samples for which in vitro LPS detection is desirable include samplesfrom a patient suspected of having a Gram-negative infection, andsamples from a product for use in a medical application (e.g., arecombinant protein solution where the protein was expressed in E.coli). Patient samples include samples of any body fluid, preferablyblood or urine. Samples may be pre-treated prior to testing by, forexample, concentrating the sample, or centrifugation to remove cells andcellular debris.

[0149] In general, in vitro detection of LPS in a sample suspected ofcontaining LPS (test sample) is performed by contacting the test samplewith an RENP of the invention for a time sufficient for the formation ofRENP-LPS complexes, and the RENP-LPS complexes detected. The RENP-LPScomplexes can be detected by virtue of a detectable label attached tothe RENP, or by the binding of an anti-LPS antibody. Binding of theanti-LPS antibody can subsequently be detected by virtue of a detectablelabel bound to the antibody, or by the binding of a detectably labeledanti-anti-LPS antibody to the RENP-LPS-antibody complex.

[0150] The in vitro assay can be performed in solution by mixing thesample with a solution containing RENP, separation of RENP-LPS complexes(e.g., by immunoprecipitation), and detection of the RENP-LPS complexesformed, e.g., by virtue of a detectable label bound to the RENP.Alternatively, the in vitro assay is performed with RENP bound to asupport, e.g., a polymeric substrate such as a microtiter well or alatex bead. Methods for binding proteins to a support are well known inthe art. For example, an anti-RENP antibody can be bound to the supportand the RENP subsequently bound to the support via binding to theanti-RENP antibody. After binding of the RENP to the support, the sampleis then contacted with the support-bound RENP and any LPS in the sampleallowed to bind to the RENP. Unbound material is then washed away, andthe RENP-LPS complexes detected by the binding of detectably labeledRENP or detectably labeled anti-LPS antibody.

[0151] The in vitro assay can also be performed as a competition bindingassay. For example, a sample suspected of containing LPS (test sample)and a known amount of detectably labeled RENP are incubated togetherwith a support having LPS bound to its surface. The test sample and theRENP may be preincubated prior to contact with the support-bound RENP.The level of detectably labeled RENP bound to the support in the testsample is compared to the level of detectably labeled RENP bound to thesupport in a negative control sample (detectably labeled RENP alone). Alevel of binding of detectably labeled RENP in the test sample which islower than binding of detectably labeled RENP in the negative controlsample is indicative of the presence of LPS in the sample.

[0152] In an alternative embodiment, the competition binding assay isperformed with support-bound RENP. In this latter assay, detectablylabeled LPS (e.g., radiolabeled LPS) is mixed with the test samplesuspected of containing LPS, and the samples contacted with thesupport-bound RENP, and the amount of detectably labeled LPS bound tothe support bound RENP detected. A level of detectably labeled LPS boundto the support in the test sample which is significantly lower than theamount of detectably labeled LPS in the negative control sample(radiolabeled LPS alone) is indicative of the presence of LPS in thetest sample.

[0153] As is apparent from the description above, the in vitro LPSassays of the invention can be performed both qualitatively andquantitatively. For example, quantitative assays can be performed bycomparing the results obtained with the test sample to results obtainedwith parallel samples containing known amounts of LPS. Quantitative invitro assays are indicative of, for example, the severity ofGram-negative infection in a patient sample from whom the sample wasobtained, or a degree of contamination where the test sample is a fluidfor administration to a patient (e.g., where the assay is performed as astep in quality control). One of ordinary skill in the art willappreciate upon reading the above-described in vitro assays thatnumerous variations of these assays can be performed without departingfrom the spirit or the scope of the invention.

[0154] Detection of LPS in vivo

[0155] Detectably labeled RENPs of the invention, preferably RENPshaving an increased LPS binding affinity relative to LBP, can be used asa diagnostic to identify a site of Gram-negative bacterial infection ina patient. For example, a detectably labeled RENP is administered to apatient suspected of having a Gram-negative infection. Preferably, thedetectable label is a radionucleotide such as ¹²³I, ¹²⁴I, ¹²⁵I, ¹²⁹I,¹³¹I, or other beta-emitting radionuclide which can be readily detectedwith either a hand-held gamma radiation detection device or by nuclearmedicine scan. Alternatively, the detectable label is a fluorescentmolecule or other visually detectable label which can be visualizedduring, for example, endoscopy. Detection can be facilitated byincreasing the ratio of detectable label to RENP.

[0156] The detectably labeled RENP is administered to the patient in anamount sufficient for binding of the RENP to the suspected infectionsite and detection of the detectable label. The detectably labeled RENPcan be administered by injection, preferably by either intravenousinjection or by direct injection into the body cavity or tissuesuspected of containing the infection site. In general, the amount ofdetectably labeled RENP administered will vary with according tonumerous variables including the RENP and detectable label used, thelocation of the suspected site of infection, the route ofadministration, and various patient factors including size, weight, age,and suspected severity of the disease.

[0157] After administration, the detectably labeled RENP is allowed tocirculate to reach the site of infection and/or incubate over thesuspected site of infection. Bound detectably labeled RENP is detectedusing methods appropriate for the label used. For example, where thedetectable label is a radionucleotide, bound RENP is detected using aradiation detecting device. Using this method, the site and the extentof a Gram-negative infection can be determined. Where desirable, thedetectably labeled RENPs can be used to label a site or sites ofinfection which can then be imaged using any of a variety of imagingtechniques known in the art (e.g., X-ray, CAT scan, MRI, or PET scan).

[0158] LPS decontamination using RENPs

[0159] The RENPs of the invention can also be used in thedecontamination of a product prior to its medical application. Forexample, where a recombinant protein has been produced by expression inE. coli, a solution containing the recombinant protein can be applied toa support having bound RENP (e.g., an affinity column). LPS in thesolution binds to the RENP bound to the support, and the LPS-freesolution is collected. If necessary, the decontamination step can berepeated multiple times until an acceptably low amount of LPS (e.g. 0 to0.001 ng/ml is detected in the solution. Such decontamination proceduresusing the RENPS of the invention can be used as a final step in qualitycontrol of, for example, recombinantly produced pharmaceuticals.

EXAMPLES

[0160] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to carry out the invention and is not intended to limit the scope ofwhat the inventors regard as their invention. Efforts have been made toensure accuracy with respect to numbers used (e.g., amounts,temperatures, etc.), but some experimental error and deviation should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1 Construction of RENPs

[0161] Specific examples of RENPs are described in Table 3, and areadditionally designated by a construct name (e.g., NCY103) or lot numberof the protein stock. TABLE 3 Examples of RENPs CONSTRUCT NAME SEQUENCEOR LOT # DESCRIPTION BPI NCY101 Native sequenceL_(1 -197(I43→ V))B_(200 → 456(N206→> D)) NCY103 LBP-BPI chimera B₁₋₁₉₉L₂₀₀₋₄₅₆ NCY104 BPI-LBP chimera B_((S351 → A)) NCY105 Glycosylationsite deleted B_((DS200 → DP)) NCY106 Formic acid cleavage site insertedL₁₋₁₉₉ B_(200-456(S351 → A)) NCY107 LBP-BPI chimera with glycosylationsite deleted B₁₋₁₉₉ NCY108 N-terminal domain of BPI B₍₁₋₁₉₀₎ Lot #159699N-terminal BPI fragment B₍₁₋₂₃₆₎ Lot #159695 N-terminal BPI fragmentB₍₁₋₂₁₂₎ Lot #159693 N-terminal BPI fragment B₁₋₁₉₉Fc NCY110 N-terminalBPI-IgG chimera B₂₀₀₋₄₅₆ NCY112 C-terminal fragment of BPI L₁₋₅₉B₆₀₋₄₅₆NCY114 LBP-BPI chimera L₁₋₁₃₄B₁₃₅₋₄₅₆ NCY115 LBP-BPI chimeraL₁₋₂₇₅B₂₇₈₋₄₅₆ NCY116 LBP-BPI chimera L₁₋₃₅₉B₃₆₀₋₄₅₆ NCY117 LBP-BPIchimera L₍₁₋₁₆₄B₍₂₀₀₋₄₅₆ Lot #164325 LBP-BPI chimera L₍₁₋₁₇₅₎B₍₂₀₀₋₄₅₆Lot #164326 LBP-BPI chimera L₁₋₁₉₇B₂₀₀₋₄₅₆ NCY118 LBP-BPI chimeraB_((F61 → C) NCY119) Cysteine insertion B_((C132 → A)) NCY120 Cysteinesubstitution B_((C132 → S)) NCY121 Cysteine substitution B_((C135 → S)NCY122 Cysteine substitution B_((C175 → S) NCY123 Cysteine substitutionB_((C132 → A)(C135 → S)(C175 → S)) NCY124 Multiple cysteine substitutionB_((1-132 → A)(C135 → S)(C175 → S)) NCY 125 Multiple cysteinesubstitution L₍₁₋₁₃₄₎B₍₁₃₆₋₃₆₁₎L₍₃₆₀₋₄₅₆₎ NCY133 LBP-BPI chimeraL₍₁₋₁₃₄₎B₍₁₃₆₋₂₇₅₎L₍₂₇₄₋₄₅₆₎ NCY134 LBP-BPI chimeraL₍₁₋₁₉₈₎B₍₂₀₂₋₂₇₅₎L₍₂₇₄₋₄₅₆₎ NCY135 LBP-BPI chimeraL₍₁₋₁₉₈₎B₍₂₀₂₋₃₆₁₎L₍₃₆₀₋₄₅₆₎ NCY136 LBP-BPI chimeraB₍₁₋₄₁₎L₍₄₂₋₁₉₉₎B₍₂₀₀₋₄₅₆₎ Lot #162303 BPI-LBP-BPI chimeraB_((1-190)(C173 → A)) Lot #162305 N-terminal BPI fragment with cationicsubstitution B_((K27 → S)(K30 → L)(K33 → T)) NCY137 Cationic Substit.(7) _((K42 → R)(K44 → P)(K48 → R)(R59 → H)) (B_(CAT7))B_((K77 → S)(K86 → R)(K90 → R)) NCY138 Cationic Substit. (6)_((K96 → S)(K118 → L)(K127 → R)) (B_(CAT6))B_((K148 → G)(K150 → D)(K160 → N)) NCY139 Cationic Substit. (9)_((K161 → Q)(R167 → Q)(K169 → V)) _((K177 → M)(K185 → D)(K198 → E))(B_(CAT9)) B_((K77 → S)(K → R)(K90 > R)) NCY140 Cationic Substit. (15)_((K96 → S)(K118 → L)(K127 → R)(K148 → G))_((K150 → D)(K160 → N)(K161 → Q)(R167 → Q))_((K169 → V)(K177 → M)(K185 → D)(K198 → E)) (B_(CAT15))L_((S77 → K)(R86 → K)(S96 → K)) NCY141 Cationic Repl. (6)_((L118 → K)(R126 → K)(L) _(CAT6)) L_((G147 → K)(D148 → K)(N158 → K))NCY142 Cationic Repl. (9) _((Q159 → K)(Q165 → R)(V167 → K)(M175 → K))_((D183 → K)(E196 → K) (L) _(CAT9))L_((S77 → K)(R86 → K)(R90 → K)(S96 → K)) NCY143 Cationic Repl. (15)_((L118 → K)(R126 → K)(G147 → K)(D148 → K))_((N158 → K)(Q159 → K)(Q165 → R)(V167 → K))_((M175 → K)(D183 → K)(E196 → K)) (L_(CAT15)) L₍₁₋₁₉₈₎B₍₂₀₁₋₄₅₆₎Fc NCY144 LBP-BPI-IgG chimera LBP NCY102 native sequence L₁₋₁₉₉ NCY109N-terminal LBP fragment L₁₋₁₉₉Fc NCY111 LBP-1g chimera L₂₀₀₋₄₅₈ NCY113C-terminal LBP fragment L_((A132 → C)) NCY126 Cysteine insertionL_((C61 → F)) NCY127 Cysteine substitution L_((C61 → S)) NCY128 Cysteinesubstitution L_((135 → S)) NCY129 Cysteine substitution L_((175 → S))NCY130 Cysteine substitution L_((C61 → F)(C135-S)(C175 → S)) NCY131Multiple cysteine substitution L_((C61 → S)(C135 → S)(C175 → S)) NCY132Multiple cysteine substitution

[0162] The proteins encoded by the LBP and L₁₋₃₅₉B₃₆₀₋₄₅₆ constructsfacilitated the LPS-mediated cellular response, indicating that LBPamino acid residues 275-359 are required for this LBP activity.

[0163] The cDNA sequences of BPI and LBP are shown in FIGS. 3A-3D and4A-C, respectively, with each nucleotide designated numerically. DNAencoding the RENPs can be prepared using a variety of techniques wellknown in the art, including protein fusion techniques, site-directedmutagenesis, and PCR (see, for example, Sambrook et al., supra; Zoller,M. J., et al., Methods Enzymol. 154:329 (1987)). For example, in theconstruction of the RENP L₁₋₁₉₇B₂₀₀₋₄₅₆, the sequence “ATAGAT₇₂₃” and“ATTGAC₇₀₀” was chosen as a convenient site to insert a ClaI restrictionsite (ATCGAT) by which to recombine portions of both BPI (former) andLBP (latter). oligonucleotide primers were designed which overlap thisregion but contain the ClaI sequence, and were synthesized on an ABI380B synthesizer (Applied Biosystems Inc., Foster City, Calif.).Additional primers were designed to bind to the 5′ and 3′-ends of bothmolecules, which primers contained NheI (5′) and XhoI (3′) restrictionsites for insertion into the vector. These primers were used to amplifyportions of the cDNA molecules encoding amino acid residues 1-199 (A)and 200-456 (B) of LBP and BPI by cyclic DNA amplification. Theresulting DNA fragments were digested with the appropriate restrictionenzymes and then purified by gel electrophoresis.

Example 2 Mammalian Expression

[0164] In order to produce BPI, LBP, or RENPs of the invention inmammalian cells, the cDNA sequences were inserted into a suitableplasmid vector. A suitable vector for such an application is pSE, whichcontains the origin of replication and early and late promoters of SV40,followed by multiple insert cloning sites, followed by the terminationsequences from the hepatitis B surface antigen gene. An origin ofbacterial DNA replication, and the genes encoding ampicillin resistanceand dihydrofolate reductase were also included in the plasmid forproduction of large amounts of DNA using bacterial host cells. Similarvectors have been used to express other foreign genes (Simonsen et al.,Biologicals 22:85 (1994). Another suitable vector, particularly forrapidly obtaining small quantities of RENPs was pCIP4 (Invitrogen Corp.,San Diego, Calif.). pCEP4 contains a CMV promoter, followed by multipleinsert cloning sites, followed by SV40 termination sequences. Alsocontained within the plasmid are an origin of bacterial DNA replication,and the genes encoding resistance to ampicillin and hygromycin B. WithpCEP4 and pSE, the same insert cloning sites as pSE for easy insertshuttling between the vectors were used. Once introduced into mammaliancell hosts, this specialized plasmid replicates as an episome, allowingsemistable amplification of introduced DNA sequences. The high gene copynumber is maintained through the selective pressure of culture in thepresence of hygromycin B.

[0165] A second expression system (EBV/293) was used to rapidly obtainsmall quantities of recombinant proteins of the subject invention whenuseful. This system was constructed to use the same insert cloning sitesas pSE for easy insert shuttling, but utilized the Epstein-Barr viruspromoter (EBV) to drive heterologous expression (pCEP4). Once introducedinto mammalian cell hosts, this specialized plasmid replicates as anepisome, allowing semistable amplification of introduced DNA sequences.The high gene copy number is maintained through the selective pressureof culture in the presence of hygromycin plus G418. Similar expressionsystems are commercially available (e.g., Invitrogen, Inc., San Diego,Calif.).

[0166] Vector DNA was prepared for acceptance of BPI cDNA by digestionwith Nhe I and Xho I, and was subsequently dephosphorylated by treatmentwith alkaline phosphatase. The prepared fragments encoding BPI, LBP, oran RENP were ligated into pSE or pCEP4, and the resulting recombinantcolonies were screened by agarose gel electrophoresis. Subsequently, theDNA sequences were confirmed by standard enzymatic sequencing methods(e.g., Sanger, 1974).

[0167] Expression plasmid DNA purified by either CsCl gradients withPlasmid or Midi Kits (Qiagen, Chatsworth, Calif.) was used to transformChinese hamster ovary strain DUXB11 (pSE) and 293-EBNA cells (InvitrogenCorp., San Diego, Calif.) (pCEP4). Transfection was performed usinglipofectin (Bethesda, Research Labs, Gaithersberg, Md.) by standardmethods. The resulting transformed cells were selected in GHT minusmedium (DUKXB11s) or in REM and 10% calf serum (293s). For the DUKXB11S,clones were selected and were passed through sequential rounds ofculture in increasing concentrations of methotrexate in order to amplifythe DHFR gene and associated heterologous genes. Supernatants fromtransfected cells, either mixed populations or clones derived from themixed population, were assayed for RENPs by ELISA using antibodiesspecific for BPI, LBP, or immunoglobulin as appropriate.

Example 3 Yeast Expression

[0168] BPI and L₁₋₁₉₇B₂₀₀₋₄₅₆ were successfully expressed in themethylotrophic yeast Pichia pastoris. Pichia was chosen as a suitableexpression system for BPI and RENPs due to its lack of LPS (endotoxin towhich BPI and RENPs bind) and its ability to produce high levels ofmammalian proteins.

[0169]Pichia pastoris strain GS115 (Invitrogen, San Diego, Calif.) wastransformed with plasmids encoding BPI and L₁₋₁₉₇B₂₀₀₋₄₅₆, andtransformed colonies were selected according to the procedures outlinedby Invitrogen (A Manual of Methods for Expression of RecombinantProteins in Pichia pastoris, Version 1.5, Invitrogen, San Diego,Calif.). For both BPI and L₁₋₁₉₇B₂₀₀₋₄₅₆, protein was secreted into themedium in a small-scale batch fermentation run. 116 ng/ml were secretedfor the one BPI construct assayed, and 14, 11, and 10 ng/ml weresecreted for the three constructs L₁₋₁₉₇B₂₀₀₋₄₅₆ constructs assayed.Secretion was assayed by enzyme-linked immunosorbant analysis (ELISA).The majority of protein for both constructs was not secreted, as shownby Western blot analysis with a polyclonal anti-BPI antibody (INVN 1-2)(prepared by conventional techniques by injecting rabbit with BPI) andalkaline phosphatase-conjugated goat anti-rabbit antibody. The Westernblot is shown in FIG. 18.

[0170] Purified BPI from Chinese Hamster ovary cells (CHOs) was used asa positive control (lane 12). In lane 1 a sample from untransformedGS115 cells served as a negative control. The antibodies did notrecognize any proteins from the untransformed GS115 cells. The nextthree lanes (2-4) were samples from colonies transformed with theconstruct for BPI and the last 6 lanes (5-10) were samples from coloniestransformed with the construct for L₁₋₁₉₇B₂₀₀₋₄₅₆. The amount ofintracellular BPI or L₁₋₁₉₇B₂₀₀₋₄₅₆ expressed in the batch fermentationrun, based on the amount of standard BPI loaded, was roughly 100 μg/mlof medium for the BPI and L₁₋₁₉₇B₂₀₀₋₄₅₆ colonies.

Example 4 Protein Purification

[0171] BPI was purified from conditioned media using the followingfour-step purification. BPI was captured on CM Sepharose (Pharmacia LKBBiotechnology). The column was washed in 50 mM Tris pH 7.4, and proteinwas eluted with 50 mM Tris buffer pH 7.4+1 M NaCl. The eluate wasdiluted 10× with 50 mM Tris pH 8.5, run over Fast Q Sepharose, and theflow-through was collected. BPI was re-captured on CM Sepharose andagain eluted as before. Buffer exchange into 10 mM Succinate+110 mM NaClpH 6 was performed using Sepharose CL₆B (Pharmacia LKB Biotechnology).Finally, Tween 20 was added to the formulated material to a finalconcentration of 0.05%.

[0172] LBP (NCY102) was captured from cell culture medium on Fast SSepharose (Pharmacia). The column was washed with 50 mM Tris pH 7.4, andprotein was eluted using 50 mM Tris pH 7.4+1 M NaCl. The eluate wasdiluted 10× in 50 mM Tris pH 8.5 and run over HiLoad Q Sepharose(Pharmacia). Protein was eluted with a 0-1 M NaCl gradient in 50 mM TrispH 8.5. Appropriate fractions were pooled according to migration on SDSPAGE electrophoresis. LBP concentration was diluted to 4.0 mg/ml, andthe pH was adjusted to 7.0 with 100 mM HCl.

[0173] L_(1-197(I43→V))B_(200-456(N206→D)) was purified from cellculture medium using the same method described for LBP.

[0174] B₁₋₁₉₉L₂₀₀₋₄₅₆ and B_((S351→A)) were purified using the sameprotocol as for BPI, except that the size exclusion step was omitted.

[0175] L₁₋₅₉B₆₀₋₄₅₆, L₁₋₁₃₄B₁₃₅₋₄₅₆ and B_(CAT6) were captured on aPoros II HS cation exchange column (PerSeptive Biosystems, Cambridge,Mass.) at pH 7.4. The column was washed with 20 mM HEPES buffer at pH7.5, and eluted with 20 mM HEPES pH 7.5 with 1 M NaCl. The eluate wasdiluted 5× in 20 mM HEPES pH 7.5 and applied to a Poros HQ anionexchange column (PerSeptive) with the flow-through applied directly to aPOROS II HS column. The POROS II HS column was eluted with 3.3 mMacetate, 3.3mM MES and 3.3 mM HEPES, pH 6.0 with a 0-1 M NaCl gradient.

[0176] L₁₋₃₅₉B₃₆₀₋₄₅₆ and L₍₁₋₁₉₈₎B₍₂₀₁₋₄₅₆₎Fc were captured fromconditioned medium at pH 7.4 on a Poros II HS column. The column waswashed with 20 mM HEPES buffer at pH 7.5, and eluted with 20 mM HEPES pH7.5+1 M NaCl. The eluate was diluted 10× with 20 mM HEPES pH 7.5, loadedon a second, smaller Poros II HS column, and eluted with 3.3 mM acetate,3.3 mM MES and 3.3 mM HEPES, pH 6 with a 0-1 M NaCl gradient.

Example 5 BPI Activity Against N. meningitidis and N. gonorrhoeae

[0177] BPI suppresses TNF release by human inflammatory cells inresponse to lipopolysaccharide (LPS) derived from a wide range ofGram-negative bacterial species. In order to test the activity of BPIagainst Gram-negative lipooligosaccharide (LOS) from the pathogenicbacteria Neisseria meningitidis and N. gonorrhoeae, non-viable bacteriawere pre-treated with recombinant BPI and incubated with human wholeblood for 4 hours at 37° C. Without BPI, N. meningitidis at 105bacteria/ml stimulated the release of 2.93±0.53 ng/ml of TNF, while N.gonorrhoeae was a more potent stimulator of TNF release: 10₄ bacteria/mlinduced 8.23±0.32 ng/ml of TNF. In both cases, 10 μg/ml BPI completelyinhibited TNF release. This indicates that BPI is able to bind anddetoxify LOS of these organisms, as well as bind LPS. Thus, BPI can beuseful as a therapeutic agent against LOS-mediated tissue damageassociated with these pathogenic Neisseria species.

Example 6 ^(biotinylated)BPI Binding Competition Assays

[0178] Competition assays for binding of LPS immobilized on microtiterplates was performed using a modified procedure described by Tobias etal., J. Biol. Chem. 264:10867 (1989). Briefly, Immulon 3 microtiterplates (96-well, Dynatech Biotechnology Products, Chantilly, Va.) werecoated with 1 or 4 μg of S. minnesota R595 Re LPS (LIST Biological Labs,Inc., #304) in 50 mM borate pH 9.5-9.8+20-25 mM EDTA overnight at 37° C.Blank, non-LPS coated wells were included on each plate and binding tothese wells was used to determine non-specific binding. Absorbancevalues from wells which were not pre-coated with LPS consistently gaveoptical density readings of less than 0.05. Plates were then washedextensively under running distilled deionized water, then dried at 37°C. Assay wells were blocked for 60 minutes at 37° C. with 1-2% very lowendotoxin BSA (Sigma, St. Louis, Mo.) prepared in pyrogen-freeTris-buffered saline (50 mM Tris pH 7.4+150 mM NaCl). The wells wereemptied, and biotinylated BPI was incubated in the presence or absenceof unlabeled BPI or recombinant protein of the subject invention dilutedin assay buffer (pyrogen-free TBS+1 mg/ml low endotoxin BSA, and 0.05%Tween-20) was incubated in the LPS coated and uncoated wells for 2-3hours at 37° C. in a total volume of 100 μl/well. After four washes inassay buffer, plates were developed with streptavidin conjugated toalkaline phosphatase (BioRad, Burlingame, Calif.) followed by 100 μl ofPNP substrate solution (Sigma) freshly prepared from two 5 mg tabletsdissolved in 10 ml substrate buffer. Substrate buffer is prepared with24.5 mg MgCl2, 48 ml diethanolamine, brought up to 400 ml, pH adjustedto 9.8 and volume brought up to 500 ml. Who Absorbances were read at 405nm on a Vmax kinetic microplate reader (Molecular Devices, Inc., MenloPark, Calif.).

[0179] The relative LPS binding affinities of BPI, LBP and RENPs weretested in the competitive binding assay described above using 10 ng/ml^(biolinylated)BPI. In these experiments, BPI inhibited^(biotinylated)BPI binding to LPS in a concentration-dependent manner(FIG. 7). Modest inhibition of ^(biotinylated)BPI-binding was observedusing NCY102 (LBP) and L_(1-197(I43→V))B_(200-456(N206→D)), suggestingthat BPI has either a higher affinity for LPS bound to a surface or thatLBP and L_(1-197(I43→V))B_(200-456(N206→D)) bind to a different site onLPS. B₁₋₁₉₉L₂₀₀₋₄₅₆, which contains the N-terminal domain of BPI,competed with ^(biatinylated)BPI at similar concentrations as unlabeledBPI, suggesting a similar affinity and binding site.

[0180] Competition between either L₁₋₁₉₇B₂₀₀₋₄₅₆ (NCY118) orL_(1-197(I43→V))B_(200-456(N206→D)) (NCY103) with biotinylated BPIoccurred at similar concentrations, giving overlapping curves (FIG. 13A)indicating that the two amino acid differences between these twomolecules [L₁₋₁₉₇B_(200-456→)L_(1-197(I43→V))B_(200-456(N206→D)):(I43→V)and (N206→D)] had no effect on affinity for immobilized LPS.L₍₁₋₁₉₈₎B₍₂₀₁₋₄₅₆₎Fc (an IgG chimera consisting of L₁₋₁₉₇B₂₀₀₋₄₅₆ linkedto human IgG1 Fc constant region of the immunoglobulin molecule) doesnot have an altered ability to compete with biotinylated BPI (FIG. 13A).L₁₋₅₉B₆₀₋₄₅₆ and L₁₋₁₃₄B₁₃₅₋₄₅₆ showed a similar affinity for LPS whichaffinity was very similar to that observed for BPI, suggesting that theregion between amino acid residues 1-59 (or 1-134) probably plays aminimal role in LPS binding (FIG. 13B). Together with data showing theB₁₋₁₉₉L₂₀₀₋₄₅₆ competes effectively with BPI (FIG. 7), these resultsindicate that amino acid residues 134-199 are important structuralcomponents of the high-affinity LPS-binding domain of BPI.

[0181] The importance of the region between amino acid residues 134 to197 in LPS affinity was further demonstrated by the markedly reducedaffinity of B_(CAT9), a mutant in which all of the cationic amino acidsof the BPI molecule (particularly the cationic residues of BPI aminoacids 134-200) are replaced with the corresponding amino acid residuesfound in LBP. These changes resulted in a molecule with binding affinityfor LPS which was more similar to that of LBP than BPI (FIG. 13C, andFIG. 7). Amino acid residues 360 to 456 of BPI are apparently notinvolved in LPS binding as demonstrated by the relative inability ofL₁₋₃₅₉B₃₆₀₋₄₅₆ to displace biotinylated BPI from LPS (FIG. 13C). Theapparent binding affinity of L₁₋₃₅₉B₃₆₀₋₄₅₆ for LPS is similar to thatof LBP and B_(CAT9), which affinity is approximately two orders ofmagnitude lower than the apparent affinity of BPI for LPS.

[0182] Thus, the domain of BPI which participates in binding toimmobilized LPS is localized in the N-terminal half of the BPI molecule,since B₁₋₁₉₉L₂₀₀₋₄₅₆ has the greatest ability to displace native BPIfrom LPS coated onto microtiter plates. This domain of BPI has been morespecifically localized to a region between amino acid residues 134-199.

Example 7 Chromogenic LAL Assay

[0183] To test the relative abilities of BPI, LBP and RENPs toneutralize LPS in vitro, these proteins were tested for inhibitoryactivity in the chromogenic LAL assay. Briefly, BPI and RENPs (25 μl of0-200 μg/ml) were pre-incubated for 1 hour at 37° C. with 1 EU/ml of E.coli 0111:B₄ LPS, (Whitaker Biologicals, Walkersville, Md.). Themixtures were then tested for LAL activity using the chromogenic LALassay kit (Whitaker Biologicals, Walkersville, Md.). The results areshown in FIG. 8 and Table 4. LPS was neutralized by the various proteinstested in the order of:B_((S351→A))≧BPI>L_(1-197(I43→V))B_(200-456(N206>D))>B₁₋₁₉₉L₂₀₀₋₄₅₆>LBP.Several studies were carried out with different lots of each protein andthe IC₅₀ values were determined. The averaged IC₅₀ values are shown inTable 4. TABLE 4 LPS Inhibition in the Chromogenic LAL Assay IC₅₀Product (μg/ml) No. of test B_((S351 → A)) 1.5 (n = 1) BPI 5.2 ± 3.3  (n= 10) L_(1-197(I43 → V))B_(200-456(N206 → D)) 28.0 ± 20.0 (n = 4)B₁₋₁₉₉L₂₀₀₋₄₅₆ 40.0 (n = 1) LBP 65.0 ± 31.0 (n = 4)

[0184] These results demonstrate that BPI neutralizes LPS activity inthe LAL assay at lower concentrations than LBP. B₁₋₁₉₉L₂₀₀₋₄₅₆, whichcontains the N-terminal domain of BPI, effectively competes with BPI forbinding to LPS (see FIG. 7) but is a relatively poor inhibitor of LPS inthe LAL assay. These results indicate that the N-terminal (LPS-binding)domain of BPI alone does not account for the neutralizing activity ofBPI in the LAL assay. L_(1-197(I43→V))B_(200-456(N206→D)) was a morepotent inhibitor than LBP or B₁₋₁₉₉L₂₀₀₋₄₅₆, suggesting that theC-terminal domain of BPI plays a very important role in endotoxinneutralization in the LAL assay.

[0185] Additional results of LPS neutralizing activity in thechromogenic LAL assay are shown in Table 5.L_(1-197(I43→V))B_(200-456(N206→D)), L₁₋₅₉B₆₋₄₅₆, and L₁₋₁₃₄B₁₃₅₋₄₅₆share the C-terminal half of the BPI molecule, again indicating thatthis domain plays an important role in LPS-neutralizing activity. Also,these data indicate that the 199-456 region is most important in LPSneutralization since adding BPI amino acid residues between 136-456 or60-456 did not improve LPS neutralizing activity. Together with the LPSbinding data, these results further indicate that the C-terminal half ofBPI is important for neutralization, while the N-terminal sequence ismore critical for LPS binding. TABLE 5 LPS Inhibition in the ChromogenicLAL Assay Protein IC50 n BPI Cumulative 1.58 ± 1.58 94 Lot #149718 1.57± 1.01 54 Lot #149719 1.69 ± 0.35 7 Lot #149722 1.70 ± 0.28 2 Lot#149724 1.41 ± 0.45 45 Lot #155794 1.95 ± 0.92 2 LBP Cumulative 55.92 ±30.53 8 Lot #151281 34.33 ± 7.45  6 Lot #151204 77.50 ± 24.45 2L_(1-197(I43 → V))B_(200-456(N206 → D)) Cumulative 22.86 ± 16.28 54 Lot#151235 25.50 ± 0.71  2 Lot #151242 36.50 ± 2.12  2 Lot #151274 3.46 ±2.18 38 Lot #159616 8.83 ± 4.91 4 B₁₋₁₉₉L₂₀₀₋₄₅₆ Cumulative 24.19 ±6.42  9 Lot #151246 12.50 ± 0.26  3 Lot #152658 10.70 1 Lot #15573740.18 ± 34.48 4 B₁₋₁₉₉ Cumulative 5.52 ± 5.05 17 Lot #151285 1.12 ± 0.002 Lot #155709 9.73 ± 1.18 3 Lot #155779 2.13 ± 0.81 2 L₁₋₅₉B₆₀₋₄₅₆ Lot#155754 3.64 ± 1.64 5 L₁₋₁₃₄B₁₃₅₋₄₅₆ Lot #155756 5.02 ± 3.11 5L₁₋₂₇₅B₂₇₈₋₄₅₆ Lot #155791 14.00 ± 2.65  3 L₁₋₃₅₉B₃₆₀₋₄₅₆ Lot#155733 >100 4 L₁₋₁₉₇B₂₀₀₋₄₅₆ Cumulative 12.75 ± 3.54  12 Lot #15575810.25 ± 30.9  8 Lot #159619 15.25 ± 5.91  4 B_(CAT6) Lot #155785 1.97 ±0.06 3 B_(CAT9) Lot #155762 29.60 ± 23.23 5 B_(CATI5) Lot #155788 7.87 ±2.80 3 L₍₁₋₁₉₈₎B₍₂₀₂₋₂₇₅₎L₍₂₇₄₋₄₅₆₎ Lot #159649 >100 3L₍₁₋₁₉₈₎B₍₂₀₁₋₄₅₆₎Fc Lot #155760 12.15 ± 6.00  4 L₁₋₁₉₉ 9.2 1 B₁₋₁₉₉10.1 ± 0.92 5 L₍₁₋₁₃₄₎B₍₁₃₆₋₂₇₅₎L₍₂₇₄₋₄₅₆₎ Lot #159643 22.00 ± 15.25 4

[0186] B_(CAT9), which contains the entire BPI sequence except for ninecationic residues between positions 148 and 197 showed very poorLPS-neutralizing activity, suggesting that these residues are importantin LPS-neutralizing activity. Similarly, this compound was relativelyineffective at competing with native BPI for binding to LPS. Thesecationic residues may permit correct structural conformation of themolecule, since both L_(1-197(I43→V))B_(200-456(N206→D)) and B_(CAT9)contain the C-terminal domain of BPI, yetL_(1-197(I43→V))B_(200-456(N206→D)) has potent neutralizing activitywhile B_(CAT9) does not.

Example 8 Inhibition of FITC-labeled LPS Binding to Human Monocytes

[0187] The relative LPs-binding affinities of RENPs of the inventionwere investigated by examining the abilities of the RENPs to inhibit LPSbinding to human peripheral blood monocytes. Blood collected in acidcitrate dextrose-containing VACUTAINER™ tubes (Becton Dickinson,Rutherford, N.J.) was diluted 1:4 in Hank's balanced salt solution(HBSS) minus calcium and magnesium (Gibco BRL, Grand Island, Md.).Mononuclear cells were isolated using Ficol-Paque (Pharmacia Inc.,Piscataway, N.J.). Cells were washed three times in HBSS, then broughtup to an appropriate volume of RPMI 1640 with glutamine and antibioticsto give approximately 1×10⁶ cells/ml. To one ml aliquots of cells,FITC-LPS was added to a final concentration of 500 ng/ml. Tubes wereclosed and incubated at 37° C. on a rocking platform. At the end of theincubation, cells were washed twice with PBS with 0.05% Human SerumAlbumin and 0.02% sodium azide. FACS analysis of the cells was performedon a FACStar flow cytometer, Immunocytometry System, Becton Dickinson(Mountain View, Calif.). The monocyte portion of the cell population wasdetermined by side scatter versus forward scatter gating and confirmedby staining a separate aliquot of cells with phycoerythrin-conjugatedanti-DR antibody (Becton Dickinson Immunocytometry Systems, Milpitas,Calif.). Results are reported as logarithmic scale mean fluorescenceintensity.

[0188] To determine the relative abilities of BPI orL_(1-197(I43→V))B_(200-456(N206→D)) to inhibit LPS binding to humanperipheral blood monocytes, isolated human peripheral blood mononuclearcells were incubated with 10% human serum containing 500 ng/mlFITC-conjugated E. coli 055:B₅ LPS in the presence or absence of BPI orL_(1-197(I43→V))B_(200-456(N206→D)). Binding of FITC-LPS to monocytescould be inhibited by increasing concentrations of both BPI andL_(1-197(I43→V))B_(200-456(N206→D)) (FIG. 9) ThusL_(1-197(I43→V))B_(200-456(N206→D)) has BPI-like activity, despite thefact that L_(1-197(I43→V))B_(200-456(N206→D)) contains the N-terminaldomain of LBP. These data, along with the results of the LPSneutralization studies shown in FIG. 8, suggest that the C-terminaldomains of BPI and LBP, and not the N-terminal domains, determinewhether the proteins inhibit or mediate LPS activation of cells.

[0189] Further studies were undertaken to determine the effects of BPI,LBP, L_(1-197(I43→V)B) _(200-456(N206→D)) and B₁₋₁₉₉L₂₀₀₋₄₅₆ onFITC-labeled LPS binding to peripheral blood monocytes in the presenceand absence of serum. In a serum-free FITC-labeled LPS binding systemwhere no LBP is available, FITC-labeled LPS does not bind to cells. Incontrast recombinant LBP facilitated LPS binding to cells atconcentrations as low as 100 ng/ml. B₁₋₁₉₉L₂₀₀₋₄₅₆ also facilitatedbinding, although to a lesser extent. Neither BPI orL_(1-197(I43→V))B_(200-456(N206→D)) promoted significant binding of LPSto cells. These data indicate that the C-terminal domain of LBP isactive in LPS binding to cells. The N-terminal domain of BPI may exertan inhibitory influence on LPS binding to cells mediated by theC-terminal domain of LBP because B₁₋₁₉₉L₂₀₀₋₄₅₆ was less active thanLBP.

[0190] Normal human serum contains about 1-10 μg/ml LBP. In the presenceof 10% autologous serum, BPI and L_(1-197(I43→V))B_(200-456(N206→D))potently inhibited FITC LPS binding to monocytes, with BPI showingslightly greater potency. B₁₋₁₉₉L₂₀₀₋₄₅₆ had marginal activity, and LBPhad no effect (FIG. 14A). These data indicate the importance of the BPIC-terminus in this test of LPS neutralization. B₁₋₁₉₉L₂₀₀₋₄₅₆, whichlacks the C-terminal domain of BPI, is approximately two orders ofmagnitude less potent at blocking LPS binding. LBP, as expected, had noeffect. Thus, BPI can intervene in the sepsis cascade by preventing LPSfrom binding to monocytes and causing release of TNFalpha.

Example 9 THP-1 Cell TNF Production Assay

[0191] THP-1 cells were obtained from the American Tissue CultureCollection (Rockville, Md.) and were maintained in REM medium containing10% fetal bovine serum, 2 mM L-glutamine, 100 units penicillin and 100μg/ml streptomycin. Cells were passed at 2×10⁵ cells/ml every 3 days.Responsiveness of THP-1 cells to LPS was induced by culturing the cellsfor 48 hours in REM medium containing 10% fetal calf serum, 2 mML-glutamine, 100 units penicillin, 100 μg/ml of streptomycin and 100 nMPMA at 37° C. in a humidified atmosphere with 5% CO₂. Cells werecultured in 96-well flat-bottomed tissue culture plates at 1-2×10⁵ cellsper well in a final volume of 200 μl. After 48 hours, adherent cellswere washed three times with 200 μl of medium without serum. To 180 μlof medium without serum but with 0.5% HSA, LPS (10 μl at 200 ng/ml)and/or BPI, LBP or other RENPs were added (10 μl at 0-2 mg/ml) and thecells were cultured for an additional 4 hours. After 4 hours,supernatants were transferred to a U-bottomed 96 well plate and theplate was centrifuged (500×g, 12 min) to pellet any cell debris.Supernatants were then stored in a second plate at −20° C. until assayedfor TNF by ELISA.

[0192] To further identify the regions of BPI which contribute toLPS-neutralizing activity, and the domains of LBP which are responsiblefor transducing the LPS signal to cells, the abilities of RENPs toreplace LBP were compared under serum-free conditions. In theseexperiments, cells of the promonocytic cell line THP-1 were induced torespond to LPS by culturing for 48 hours with phorbol ester. Afterinduction, cells were stimulated with 19 ng/ml of LPS in the presence orabsence of the recombinant protein. In this system, TNF release requiresa source of LBP. Data from these experiments (FIG. 15) show that onlyLBP and L₁₋₃₅₉B₃₆₀₋₄₅₆ stimulated TNF release. Thus the domain of LBPresponsible for facilitating LPS-induced TNF release is within aminoacid residues 199-359. Interestingly, B₁₋₁₉₉L₂₀₀₋₄₅₆ did not mediate TNFrelease in a serum-free system. This may indicate that the N-terminaldomain of BPI binds too tightly to LPS to allow transfer of LPS to CD14on the 1.5 macrophage surface. FIG. 16 shows an additional comparison ofTNF production. Because L₍₁₋₁₉₈₎B₍₂₀₂₋₂₇₅₎L₍₂₇₄₋₄₅₆₎ includes LBP domain274-456 and has activity, the active domain may comprise only residues274-359.

Example 10 LPS-Induced TNF Release In Whole Blood

[0193] Peripheral blood from normal human volunteers was collected inheparin-containing VACUTAINER™ tubes (Becton Dickinson). To onemilliliter of whole blood, BPI, a protein of the subject invention, orbuffer control was added, followed by 1 ng/ml E. coli 055:B₅ refinedstandard endotoxin (RSE) (Whitaker Bioproducts). Samples were incubatedin closed microtubes at 37° C. for 4 hours on a rocking platform. At theend of the incubation, samples were centrifuged for 5 minutes at 500×gat 4° C., the plasma collected and frozen on dry ice until assayed forthe presence of cytokines. TNF levels were determined by ELISA usinghuman recombinant TNF (Genentech Inc., South San Francisco, Calif.) as astandard.

[0194] In later studies it was determined that BPI activity in wholeblood is inhibited by heparin, and the anticoagulant was changed tocitrate. In these experiments, to 120 μl of citrated whole blood, 20 μlof BPI or an RENP (at 0-1 mg/ml) or buffer control, 20 μl of 100 ng/mlof E. coli 055:B₅ LPS was added to stimulate cells in whole bloodsamples. These experiments were performed in polypropylene microtiterplates (Costar, Cambridge, Mass.). After the 37° C. incubation step, theplates were centrifuged 15 min at 500×g at 4° C. and the plasma removedfor assaying.

[0195] To test the effects of BPI, LBP, and RENPs on LPS activation ofTNF production in whole blood, BPI, LBP,L_(1-197(I43→V))B_(200-456(N206->D)), or B₁₋₁₉₉L₂₀₀₋₄₅₆ was mixed withheparinized blood, and LPS was added to the resulting mixture. The bloodwas incubated for four hours at 37° C., and TNF in the plasma wasmeasured as described, supra. to Results are shown in FIG. 10.L_(1-197(I43→V))B_(200-456(N206→D)) was the most potent at blocking TNFrelease, followed by BPI as the next most potent blocker. B₁₋₁₉₉L₂₀₀₋₄₅₆and LBP had essentially no effect. Thus, in whole blood,L_(1-197(I43→V))B_(200-456(N206→D)) proved to be the most potentinhibitor of LPS-mediated cytokine stimulation.

[0196] When experiments were performed in citrated rather thanheparinized whole blood, endotoxin-neutralizing activity of BPI andL_(1-197(I43→V))B_(200-456(N206→D)) were equivalent (Table 6). Inexperiments in which recombinant proteins were preincubated withendotoxin before addition to whole blood, the activities of thesecompounds fell roughly into two groups. BPI,L_(1-197(I43→V))B_(200-456(N206→D)), B₁₋₁₉₉, B_(CAT6), B_(CAT15),L₁₋₅₉B₆₀₋₄₅₆, L₁₋₁₃₄B₁₃₅₋₄₅₆, and L₁₋₁₉₇B₂₀₀₋₄₅₆ possessLPS-neutralizing activity, while LBP, B₁₋₁₉₉L₂₀₀₋₄₅₆, L₁₋₁₉₉,L₁₋₃₅₉B₃₆₀₋₄₅₆ and B_(CAT9) were relatively inactive. Results withL₁₋₂₇₅B₂₇₈₋₄₅₆, B_(CAT9), and L₍₁₋₁₉₈₎B₍₂₀₁₋₄₅₆₎Fc were equivocal. Whencompounds were added to the blood samples immediately prior to LPS, theIC50 values were higher, but the same group of proteins showed activity.These data further indicate the role of the C-terminal region of BPI,demarcated by amino acid residues 200-359, in LPS neutralization in aphysiological environment such as whole blood. Because L₁₋₁₉₉ is not apotent endotoxin-neutralizing protein (see Tables 9 and 11), it can beconcluded that the C-terminal domain of BPI must significantlycontribute to the endotoxin-neutralizing activity ofL_(1-197(I43→V))B_(200-456(N206→D)) and L₁₋₁₉₇B₂₀₀₋₄₅₆. All compoundswhich contain this sequence (201-359) are active except B_(CAT9), whichwas also inactive in other assays possibly because the cationic aminoacid residues which were replaced may be important in configuring themolecule. These data indicate that L_(1-197(I43→V))B_(200-456(N206->D))is equivalent to L₁₋₁₉₇B₂₀₀₋₄₅₆ in activity, thus implying that theamino acid differences between these two proteins have no affect uponLPS binding affinity. TABLE 6 LPS Inhibition in Human Whole BloodProtein Not Pre- IC50 Pre- IC50 incubated (μg/ml) n incubated (μg/ml) aL₁₋₁₃₄B₁₃₅₋₄₅₆ 0.15 ± 0.12 3 BPI 2.60 ± 1.52 5 L₁₋₁₉₇B₂₀₀₋₄₅₆ 2.90 ±3.59 12 L₁₋₁₃₄B₁₃₄₋₄₅₆  3.7 ± 1.60 2 L₁₋₅₉B₆₀₋₄₅₆ 0.28 ± 0.25 3L_(1-199\7(I43−>V))B_(200-456(N206−>D)) 7.13 ± 5.19 4L_(1-197(I43−>V))B_(200-456(N206−>D)) 0.16 ± 0.11 17 L₁₋₅₉B₆₀₋₄₅₆   15 ±18.58 2 BPI 0.43 ± 0.49 13 L₁₋₁₉₇B₂₀₀₋₄₅₆ 26.5 ± 0.71 2L₍₁₋₁₉₈₎B₂₀₁₋₄₅₆₎Fc 18.00 ± 27.73 3 L₁₋₃₅₉B₃₆₀₋₄₅₆ >100 1B₁₋₁₉₉L₂₀₀₋₄₅₆ >100 3 B_(CAT9) >100 2 L₁₋₃₅₉B₃₆₀₋₄₅₆ >100 3L₍₁₋₁₉₈₎B₍₂₀₁₋₄₅₆₎FC >100 2 B_(CAT 9) 11.50 ± 3.54  2* B₁₋₁₉₉L₂₀₀₋₄₅₆ NDB₁₋₁₉₉ 0.73 ± 0.48 6 B₁₋₁₉₉ 4.0 1 L₁₋₁₉₉ >100 2 L₁₋₁₉₉ >100 1 B_(CAT15)0.21 ± 0.26 3 B_(CAT6) 0.27 ± 0.25 2 L₍₁₋₁₃₄₎B₍₁₃₆₋₂₇₅₎L₍₂₇₄₋₄₅₆₎ 2.0 1L₍₁₋₁₉₈₎B₍₂₀₂₋₂₇₅₎L₍₂₇₄₋₄₅₆₎ 5.27 ± 5.83 3 L₁₋₂₇₅B₂₇₈₋₄₅₆ 38.10 ± 53.643

Example 11 Mouse Serum Half-Life Assay

[0197] CD-1 mice weighing approximately 20 grams were injected with 5mg/kg body weight BPI, LBP, or RENPs (1 mg/ml) at time zero. Inheparinized (or later EDTA-containing) tubes, blood was collected fromthe retroorbital plexus from three animals for each time point tested. Atypical blood collection schedule was 5, 10, 15, 30, 45, 60, 90, 120,240, and 360 minutes. The EDTA anticoagulated blood was centrifuged forabout 10 min at 1000×g and the supernatant plasma removed and storedfrozen on dry ice until tested. Levels of BPI, LBP, or RENP in theplasma samples were determined by ELISA using the appropriate protein asthe standard.

[0198] A potent anti-endotoxin therapeutic should not only neutralizeendotoxin, but should also have the capacity to clear endotoxin from thecirculation. The circulating levels of radioactively labeled ¹²⁵I-BPIwere measured in the mouse in the presence and absence of endotoxin(Table 7). In the absence of endotoxin, the elimination (alpha) phasefor ¹²⁵I-BPI was less than two minutes. In the presence of LPS, thealpha phase was extended to 6.2 minutes. ¹²⁵I-LPS alone has a singlephase distribution (beta) with a half-life of about 101 minutes. When¹²⁵I-LPS and unlabeled BPI were administered, a 6.2 minute elimination(alpha) phase was observed, indicating that elimination was remarkablyfacilitated by BPI. TABLE 7 Serum Half-Life of BPI and LPS in the MouseTest Article t_(1/2)alpha t_(1/2)beta ^(125I-BPI) 1.6 103.0^(125I—BPI + LPS) 6.3 72.0 ^(125I—LPS) 101.0 ^(125I—LPS + BPI) 6.2 114.0

[0199] In order to determine whether the very short circulatinghalf-life of BPI could be extended by molecular engineering, thecirculating half-lives of BPI, LBP, B₁₋₁₉₉L₂₀₀₋₄₅₆ andL_(1-197(I43→V))B_(200-456(N206→D)) were compared (FIG. 11). Using bothlabeled and unlabeled material, it was observed that the circulatinghalf-life of BPI in the mouse is remarkably short. This may be a resultof the highly cationic nature of BPI having a predicted pI of 10.6. LBP,normally present in the circulation at concentrations of 10 μg/ml, has apredicted pI of about 6.8. As expected,L_(1-197(I43→V))B_(200-456(N206>D)) (LBP-BPI chimera lacking BPIcationic residues) has a markedly longer circulating half-life thanB₁₋₁₉₉L₂₀₀₋₄₅₆ (BPI-LBP chimera having BPI cationic residues). FIG. 11shows that L_(1-197(I43→V))B_(200-456(N206→D)) indeed has a longerhalf-life than BPI. B₁₋₁₉₉L₂₀₀₋₄₅₆, with the N-terminal domain of BPI,had an even shorter half-life than that of BPI. Thus, the N-terminaldomain of BPI appears to play a major role in its short circulatinghalf-life.

[0200] Further pharmacokinetic studies were performed in whichrecombinant proteins of the subject invention were administered to CD-1mice at a 5 mg/kg bolus dose. Results of these experiments are shown inFIGS. 17A-17H. At 5 mg/kg, the circulating half life of B₁₋₁₉₉L₂₀₀₋₄₅₆was similar to that of BPI. L_(1-197(I43→V))B_(200-456(N206→D)) andL₁₋₁₉₇B₂₀₀₋₄₅₆ had overlapping elimination curves and again indicatingthat these two molecules are equivalent with respect to their biologicalactivities. L_(1-197(I43→V))B_(200-456(N206→D)) and B₁₋₁₉₉ persisted inthe circulation significantly longer than BPI or B₁₋₁₉₉L₂₀₀₋₄₅₆, but notas long as the serum protein LBP. Comparison of the elimination curvesof L₁₋₅₉B₆₀₋₄₅₆, L₁₋₁₃₄B₁₃₅₋₄₅₆ and B_(CAT9) revealed that theN-terminus of LBP plays a role in extending circulating half-life.L₁₋₅₉B₆₀₋₄₅₆ circulates slightly longer than BPI, and contains the leastLBP sequence of any of the recombinant proteins tested (amino acidresidues 1-59). L₁₋₁₃₄B₁₃₅₋₄₅₆ was cleared somewhat more slowly,indicating that LBP amino acid residues 60-134 of LBP impart a longercirculating half-life. In contrast, the cationic residues of BPI between134-199 shorten the half-life, since in B_(CAT9), where the cationicresidues in this region were replaced with the corresponding residues ofLBP, the half-life was similar to that of L₁₋₁₃₄B₁₃₅₋₄₅₆. Including moreLBP residues in the N-terminal domain further extends the half life. Ifamino acid residues 199-359 of LBP are added (L₁₋₃₅₉B₃₆₀₋₄₅₆), thehalf-life is longer, but not quite as long as that of LBP. LikewiseL₍₁₋₁₉₈₎B₍₂₀₂₋₂₇₅₎L₍₂₇₄₋₄₅₆₎ (with LBP domain 1-198 and 274-456) has arelatively long t½. These results indicate that the more “LBP-like” themolecule is, the longer it circulates. In addition, combining an Igfragment Fc with L_(1-197(I43→V))B_(200-456(N206→D)) gives the longesthalf life.

Example 13 Mouse Endotoxin Challenge Assay

[0201] Female CD-1 mice were injected in the lateral tail vein with aLD₁₀₀ dose (25-35 mg/kg) of Salmonella abortus equi endotoxin, which wasfollowed by an injection of BPI, RENP, or vehicle control into theopposite lateral tail vein at the indicated time. Protein injectionconcentrations varied and provided doses of 0.5, 1, and 5 mg/kg. Use ofvehicle control illustrated the lethality of the endotoxin challenge inthe test animal. Animals were observed for mortality at 24, 28, and 72hours. Preliminary studies showed that mortality does not change fromday three to day seven or beyond.

[0202] The efficacies of BPI, LBP, L_(1-197(I43→V))B_(200-456(N206→D)),B₁₋₁₉₉L₂₀₀₋₄₅₆ and B_((S351→A)) against lethal endotoxin challenge inmice were compared (Tables 8-10). The efficacies ofL_(1-197(I43→V))B_(200-456(N206→D)), L₁₋₁₉₇B₂₀₀₋₄₅₆, L₁₋₅₉B₆₀₋₄₅₆,L₁₋₁₃₄B₁₃₅₋₄₅₆, L(₁₋₁₉₈₎B₍₂₀₁₋₄₅₆₎Fc, L₁₋₂₇₅B₂₇₈₋₄₅₆, L₁₋₃₅₉B₃₆₀₋₄₅₆,B_(CAT9), B_(CAT6), and B_(CAT15) against lethal endotoxin challenge inmice were also compared (Table 11). When each protein was given withintwo minutes after lethal endotoxin challenge, BPI,L_(1-197(I43→V))B_(200-456(N206→D)) and B_((S351→A)) had similarpotency, whereas LBP and B₁₋₁₉₉L₂₀₀₋₄₅₆ showed minimal protection. Themarginal protective effects of LBP and B₁₋₁₉₉L₂₀₀₋₄₅₆ since these agentsdo not block the inflammatory signal of LPS acting on human cells invitro (FIG. 10). TABLE 8 Mouse Endotoxin Challenge Comparison of BPI,LBP (NCY102). and L_(1-197(143-V))B_(200-456(N2O6 → D)) (NCY103) DrugDose % Survival (n = 10) Control 0 mg/kg  0% BPI 5 mg/kg 60% 1 mg/kg 40%LBP 5 mg/kg 30% 1 mg/kg 20% L_(1-197(143 → V))B_(200 → 456(N206 → D)) 5mg/kg 60% 1 mg/kg 50%

[0203] TABLE 9 Mouse Endotoxin Challenge Comparison of BPI,L_(1 − 197(I43−> V)) B_(200 − 456(N206−> D)) and B_((S351−> A)) DrugDose % Survival (n= 10) Control 0 mg/kg  0% BPI 5 mg/kg 80%L_(1 − 197(I43−> V))B_(200 − 456(N206−> D)) 5 mg/kg 100% B_((S351−> A))5 mg/kg 90%

[0204] TABLE10 Mouse Endotoxin Challenge Comparison of BPI andB₁₋₁₉₉L₂₀₀₋₄₅₆(NCY104) Drug Dose % Survival (n = 10) Control   0 mg/kg 40% BPI  10 mg/kg 100%   2 mg/kg 100% 0.4 mg/kg 70% B₁₋₁₉₉L₂₀₀₋₄₅₆  10mg/kg  60%   2 mg/kg  60% 0.2 mg/kg 50%

[0205] TABLE 11 Survival in CD-1 Mice Following Lethal EndotoxinChallenge Panel A % p Survivors/n Survival (vs. control) BPI 40/50 80.00 <0.001 L_(1-197(I43 −> V))B_(200-456(N206 −> D)) 17/20  85.00<0.001 L₁₋₁₉₇B₂₀₀₋₄₅₆ 16/20  80.00 <0.001 L₁₋₅₉B₆₀₋₄₅₆ 13/20  65.00<0.001 L₁₋₁₃₄B₁₃₅₋₄₅₆ 13/20  65.00 <0.001 L₍₁₋₁₉₈₎B₍₂₀₁₋₄₅₆₎Fc 5/1050.00 0.002 L₁₋₃₅₉B₃₆₀₋₄₅₆ 2/10 20.00 0.149 B_(CAT6) 9/10 90.00 <0.001B_(CAT9) 1/10 10.00 0.442 L₁₋₂₇₅B₂₇₈₋₄₅₆ 0/10 0 — B_(CAT15) 6/10 60.0<0.05 Control 1/30 3.30 —

[0206] Panel B Dose Survivors % p mg/kg (n = 20) Survival (vs. control)*BPI 5 13 65 <0.001 1 9 45 0.001 0.5 6 30 0.02L_(1-197(I43 −> V))B_(200-456(N206 −> D)) 5 18 90 <0.001 1 12 60 <0.0010.5 9 45 0.001 B₁₋₁₉₉ 5 3 15 NS 1 0 0 NS 0.5 1 5 NS

[0207] L_(1-197(I43→V))B_(200-456(N206→D)) was markedly more effectivethan BPI when given more than an hour before or after LPS (FIG. 12).These results indicate that the longer circulating half-life ofL_(1-197(I43→V))B_(200-456(N206→D)), or perhaps the increased ability ofL_(1-197(I43→V))B_(200-456(N206→D)) to inhibit endotoxin in whole blood,has a dramatic effect on L_(1-197(I43→V))B_(200-456(N206→D)) efficacy invivo.

[0208] Further experiments were performed to assess the LPS-neutralizingactivities of recombinant proteins of the subject invention in vivo. Inthese experiments, a lethal LPS challenge was administered at time zero,followed immediately by a 5 mg/kg bolus injection of recombinantprotein.

Example 12 BPI Reduction of LPS-Induced Cytokine Function and Mortalityin Rats

[0209] The potential effect of BPI against LPS related cytokineformation and mortality was investigated in rats with either (a)hemorrhagic shock (bled to lower pressure to 30-35 mmHg mean arterialpressure for 90 minutes, followed by reinfusion of shed blood and anequal volume of Ringer's over 30 minutes), or (b) endotoxic shock(caused by 100 μg LPS and 500 mg D-galactosamine/ kg). Similarly,recombinant BPI binds LPS and inhibits TNF formation in vitro. Treatmentcomprised 5 mg BPI/kg i.v. for the BPI group, or 1 ml saline i.v. forthe control group.

[0210] The results of the investigation of BPI efficacy in rats witheither (a) hemorrhagic shock or (b) endotoxic shock show that (a) inrats with hemorrhagic shock, the mortality was decreased from {fraction(5/10)} (50% control group) to {fraction (2/10)} (20% BPI group) at 48hours; (b) in rats with endotoxic shock, the 5-day mortality wassignificantly reduced (p=0.055) by BPI treatment to 43%, as compared to83% in the control group. Plasma LPS levels were at least partiallyneutralized at two hours (5.9±4.1 vs 10.8±4.1 ng/ml). Cytokine formationwas concomitantly reduced in the BPI group as measured by plasma TNFlevels at two hours (3.9±2.9 vs 10.3±6.3 ng/ml). Liver transaminases(GOT and GPT, whose elevation indicates hepatic dysfunction) andbilirubin still increased at eight hours; however, the increase was lesswith BPI. These data demonstrate that BPI has utility as a therapeuticagent against endotoxin-related disorders in hemorrhagic as well asendotoxic shock.

Example 14 Protection Against LPS Challenge by Intrapulmonary Deliveryof RENPs

[0211] Anesthetized male CD-1 mice were treated intra-nasally with 1 or10 μg of either BPI or L_(1-197(I43→V))B_(200-456(N206→D)) in 50 μl.Control animals received 50 μl of saline for injection. After 20minutes, animals were re-anesthetized, and challenged with 10 ng of E.coli 055:B₅ LPS. One hour after endotoxin challenge, mice werere-anesthetized, and 0.7 ml of saline containing 1% human serum albuminwas added to the lungs via the trachea. The lungs were gently kneaded. A0.5 ml volume of BAL (bronchoalveolar lavage) fluid was aspirated, cellswere pelleted by centrifugation, and the BAL sample was sorted at −70°C. The TNF-alpha level in the BAL fluid was determined by ELISA (resultsshown in FIG. 19).

[0212]FIG. 19 shows that endotoxin-neutralizing proteins such as BPI andL_(1-197(I43→V))B_(200-456(N206→D)) (NCY103) can also neutralizeendotoxin-mediated TNF release in the lung. These results indicate thatthese proteins are effective when delivered directly into the lung andthus may be useful for treatment of pneumonias and otherendotoxin-related disorders of the lung, such as ARDS.

Example 15 Construction of L₁₋₁₉₇B₂₀₀₋₄₅₆

[0213] cDNA encoding L₁₋₁₉₇B₂₀₀₋₄₅₆ was constructed by creating a uniqueClaI site at the junction between the nucleotide sequence coding forIle₁₉₇-Asp₁₉₈ residues (ATA-GAT→ATC-GAT). For L₁₋₁₉₇B₂₀₀₋₄₅₆, a 0.7 kbNheI/ClaI DNA fragment (encoding amino acids 1-197) derived from the 5′sequence of LBP and a 0.8 kb ClaI/XhoI fragment (encoding amino acids200-456) derived from the 3′ sequence of BPI were generated by PCR. Thechimeric cDNAs were spliced together by cloning the fragments into pSE,a mammalian vector. The cDNAs for BPI, LBP and L₁₋₁₉₇B₂₀₀₋₄₅₆ weretransfected into Chinese hamster ovary cells (strain DUXB11) usinglipofectin. The resulting transformed cells were selected, andexpression was amplified with methotrexate. Cell culture supernatantswere screened for reactivity by ELISA. Recombinant BPI, LBP, andL₁₋₁₉₇B₂₀₀₋₄₅₆ were purified as described above.

Example 16 Pharmokinetics of L₁₋₁₉₇B₂₀₀₋₄₅₆

[0214] Data for pharmacokinetic analysis were collected from healthyCD-1 mice given a single bolus injection (5 mg/kg) of recombinantprotein at time=0. Blood was collected from three mice for eachcollection time point by retroorbital puncture at timepoints over threehours. Blood samples anticoagulated in EDTA were assayed by a doubleantibody sandwich ELISA for the presence of BPI, LBP or L₁₋₁₉₇B₂₀₀₋₄₅₆).Pharmacokinetic analysis was performed using a non-compartmentalanalysis (PharmK pharmacokinetic software, SoftRes, Inc.).

[0215] Comparison of BPI and LBP shows that BPI was cleared rapidly witha clearance rate of 13.0 ml/minute (Table 12). LBP had the longest halflife, with a clearance rate of 0.042 ml/min. Compared to BPI, LBP wascleared 310 times more slowly. L₁₋₁₉₇B₂₀₀₋₄₅₆ had an intermediate halflife (Clearance rate=0.175 ml/min), being cleared 74 times more slowlythan BPI. TABLE 12 Clearance rate of L₁₋₁₉₇B₂₀₀₋₄₅₆ CL (ml/min) (vs.BPI) BPI 13.000 — LBP 0.042 (310 fold) L₁₋₁₉₇B₂₀₀₋₄₅₆ 0.175  (74 fold)

Example 17 LPS Protection by L₁₋₁₉₇B₂₀₀₋₄₅₆

[0216] Female CD-1 mice (n=10) were injected in the lateral tail veinwith 35 mg/kg S. abortus equi LPS (Sigma, St. Louis, Mo.) at time=0.Recombinant protein (5 mg/kg) was then administered intravenously intothe opposite lateral tail vein immediately following (t=0) endotoxinchallenge. Survival was monitored at 24, 48 and 72 hours post-challenge.Control animals received 0.1 ml saline instead of recombinant protein.The p values were determined by Fisher's exact test.

[0217] The results are shown in FIG. 20. BPI and L₁₋₁₉₇B₂₀₀₋₄₅₆ provided90% to 100% survival, respectively, at the 72 hour end point. No furthermortality was noted at seven days post-challenge. The untreated controlgroup had a survival rate of 20%. The survival rates of the treatedgroups were statistically significant compared to the control group(p<.001 for the L₁₋₁₉₇B₂₀₀₋₄₅₆ group and p=.003 for the BPI groupdetermined by Fisher's exact test). These results indicate thatL₁₋₁₉₇B₂₀₀₋₄₅₆ is as effective as BPI in this endotoxin challenge modelin vivo.

Example 18 Protection Against Endotoxin Challenge in Mice

[0218] The ability of the recombinant, endotoxin-neutralizing proteinsB₍₁₋₄₁₎L₍₁₋₁₉₉₎B₍₁₋₄₅₆₎, L₍₁₋₁₆₄₎B₍₂₀₀₋₄₅₆₎, B₍₁₋₁₇₅₎B₍₂₀₀₋₄₅₆₎,B₍₁₋₂₃₆₎, and B₍₁₋₁₉₀₎ to protect mice against endotoxin challenge wascarried out as described in Example 17 above. Protection by theseproteins was compared to the protection provided by BPI or saline. Theresults of these studies are shown in Table 13. TABLE 13 Number ofSurvivors/10 at Time (hours) Compound Lot # 0 12 18 24 36 48 60 72 Group1 native BPI 149724 10 10 10 10 10 9 9 9 Group 2 B₍₁₋₄₁₎L₍₁₋₁₉₉₎B₍₁₋₄₅₆₎162303 10 10 10 10 9 9 8 8 Group 3 L₍₁₋₁₆₄₎B₍₂₀₀₋₄₅₆₎ 164325 10 10 9 9 88 7 7 Group 4 L₍₁₋₁₇₅₎B₍₂₀₀₋₄₅₆₎ 164326 10 10 10 10 10 10 10 10 Group 5B₍₁₋₂₃₆₎ 159695 8 7 5 4 1 0 0 0 Group 6 B₍₁₋₁₉₀₎ 159699 10 9 8 6 6 6 5 5Group 7 Saline 10 8 7 6 4 3 3 3

Example 19 Detection of a Gram-negative Infection in a Patient

[0219] A blood sample of about 1 ml to 5 ml is drawn from a patientsuspected of having a Gram-negative infection. The blood sample istreated with citrate anti-coagulant and plasma is separated from theblood cells by centrifugation. The plasma is then diluted in a series of10-fold dilutions in assay buffer (pyrogen-free TBS+1 mg/ml lowendotoxin BSA, and 0.05% Tween-20). The diluted plasma samples are thenmixed with a known amount of biotinylated RENP. A series of controlsamples containing known amounts of biotinylated RENP in assay buffer isincluded in the assay as quantitative and negative controls.

[0220] The test and control samples are then applied to the wells of amicrotiter plate having bound LPS. The LPS-bound microtiter wells areprepared by incubation with 1 or 4 μg of S. minnesota R595 Re LPS (LISTBiological Labs, Inc., #304) in 50 mM borate pH 9.5-9.8+20-25 mM EDTAovernight at 37° C. Blank, non-LPS coated wells are included on eachplate as controls for non-specific binding. The plates are then washedextensively under running distilled deionized water, then dried at 37°C. The assay wells are subsequently blocked for 60 minutes at 37° C.with 1-2% very low endotoxin BSA (Sigma, St. Louis, Mo.) prepared inpyrogen-free Tris-buffered saline (50 mM Tris pH 7.4+150 mM NaCl).

[0221] The test and control samples are incubated for a time sufficientfor binding of the RENP in the samples to the LPS bound to themicrotiter wells, generally about 2-3 hours at 37° C. in a total volumeof 100 μl/well. After incubation, the wells are washed four times withassay buffer, and the plates are developed with streptavidin conjugatedto alkaline phosphatase followed by 100 μl of PNP substrate solutionfreshly prepared from two 5 mg tablets dissolved in 10 ml substratebuffer. Substrate buffer is prepared with 24.5 mg MgCl₂, 48 mldiethanolamine, brought up to 400 ml, pH adjusted to 9.8 and volumebrought up to 500 ml. Absorbances are read at 405 nm on a microplatereader.

[0222] If the level of biotinylated RENP bound to the wells of the testsample is significantly less than the level of biotinylated RENP boundto the negative control sample, then the patient has endotoxincirculating in the bloodstream which is generally associated with aGram-negative infection. Moreover, the level of RENP binding in the testsample is compared to the levels of RENP binding in the quantitativecontrols, each of which are representative of varying degrees ofseverity of Gram-negative infection in a patient. The level of bindingof the test sample is thus compared to the levels of binding of thequantitative samples to determine a degree of severity of infection.

Example 20 Detection of a Gram-negative Infection In Vivo

[0223] RENP is detectably labeled with ¹²⁵I using methods well known inthe art. Approximately 100 μg of an ¹²⁵ I-labeled RENP is injectedintravenously into a patient suspected of having a Gram-negativeinfection in an organ, e.g., the liver. After allowing a time sufficientfor circulation of the ¹²⁵I-labeled RENP to the suspected site ofinfection, the abdomen of the patient is fluoroscoped or X-rayed 2 to 3times so as to include various perspectives. The X-ray is then examinedto identify sites of binding of the RENP by virtue of an abnormallydarkened section on the X-ray. Upon identification of the site ofinfection, the clinician designs an appropriate therapeutic regimen.

[0224] Following procedures similar to those described above, otherrecombinant, LPS-binding proteins can be produced and used in diagnosticmethods and methods of treatment according to the invention.

[0225] The invention now being fully described, it will be apparent toone of ordinary skill in the art that many changes and modifications canbe made thereto without departing from the spirit or scope of theappended claims.

What is claimed is:
 1. A method of detecting a site of Gram-negativebacterial infection in a subject, said method comprising the steps of:injecting into the patient's circulatory system an injectableformulation comprising an effective amount of a recombinantendotoxin-neutralizing polypeptide attached to a detectable label,wherein the polypeptide is characterized by (i) selective and specificbinding to lipopolysaccharide and (ii) endotoxin-neutralizing activity,with the proviso that the amino acid sequence of the polypeptide is notidentical to the amino acid sequences of BPI or LBP; allowing thedetectably labeled polypeptide sufficient time to circulate in thesubject and bind to lipopolysaccharide in the patient; and detecting asite of label binding in the patient, thereby detecting a site ofGram-negative bacterial infection.
 2. The method of claim 1, wherein thepolypeptide is covalently bound to a molecule which enhances thehalf-life of the polypeptide.
 3. The method of claim 1, wherein thepolypeptide contains an LPS binding domain of BPI, LBP, a BPI variant,or an LBP variant.
 4. The method of claim 1, wherein the detectablelabel is a radionucleotide.
 5. A method of detecting a Gram-negativebacterial infection in a subject, said method comprising the steps of:obtaining a sample from a patient suspected of having a Gram-negativebacterial infection; contacting said sample with a detectably labeledrecombinant endotoxin-neutralizing polypeptide for a time sufficient forbinding of the polypeptide to lipopolysaccharide in the sample; anddetecting formation of lipopolysaccharide-polypeptide complexes bydetection of a detectable label bound to the polypeptide; whereindetection of a level of detectable label in said sample significantlygreater than a level of detectable label in a negative control sample isindicative of a Gram-negative bacterial infection in the subject.
 6. Themethod of claim 5, wherein the polypeptide contains an LPS bindingdomain of BPI, LBP, a BPI variant, or an LBP variant.
 7. The method ofclaim 5, wherein said detection is quantitative.
 8. The method of claim7, wherein said quantitative detection is correlated with anGram-negative bacterial infection load.
 9. A detectably labeledrecombinant endotoxin-neutralizing polypeptide characterized by (i)selective and specific binding to lipopolysaccharide and (ii)endotoxin-neutralizing activity, with the proviso that the amino acidsequence of the polypeptide is not identical to the amino acid sequencesof BPI or LBP.
 10. A polypeptide according to claim 9, wherein thepolypeptide contains an LPS binding domain of BPI, LBP, a BPI variant,or an LBP variant.
 11. A detectably labeled polypeptide according toclaim 9, wherein the polypeptide comprises a molecule which enhances thehalf-life of said polypeptide and is covalently bound to thepolypeptide.
 12. A detectably labeled polypeptide according to claim 11,wherein said molecule is an immunoglobulin fragment, a half-lifeenhancing porion of LBP, a half-life enhancing portion of an LBPvariant, or polyethylene glycol.
 13. A kit for detecting a site ofGram-negative bacterial infection in a subject, the kit comprising: aninjectable formulation comprising a detectably labeled recombinantendotoxin-neutralizing polypeptide characterized by (i) selective andspecific binding to lipopolysaccharide and (ii) endotoxin-neutralizingactivity, with the proviso that the amino acid sequence of thepolypeptide is not identical to the amino acid sequences of BPI or LBP.14. A recombinant endotoxin-neutralizing polypeptide characterized by(i) selective and specific binding to lipopolysaccharide and (ii)endotoxin-neutralizing activity, with the proviso that the amino acidsequence of the polypeptide is not identical to the amino acid sequencesof BPI or LBP.
 15. A recombinant endotoxin-neutralizing polypeptideaccording to claim 14, wherein said polypeptide is of the formulaL₁₋₁₉₇B₂₀₀₋₄₅₆ or a corresponding protein which (a) functions to bindlipopolysaccharide and (b) neutralizes endotoxin.
 16. A recombinantendotoxin-neutralizing polypeptide according to claim 14, wherein thepolypeptide is BPI(S351→X), wherein X is any amino acid other thanserine.
 17. A recombinant endotoxin-neutralizing polypeptide accordingto claim 16, wherein X is alanine.
 18. A recombinantendotoxin-neutralizing polypeptide according to claim 14, wherein thepolypeptide contains the amino acid sequence of BPI having a cationicamino acid substituted with a neutral or anionic residue.
 19. Arecombinant endotoxin-neutralizing polypeptide according to claim 18,wherein the cationic amino acid is at BPI amino acid residue positions27, 30, 33, 42, 44, 48, 59, 77, 86, 90, 96, 118, 127, 148, 150, 160,161, 167, 169, 177, 185, or
 198. 20. A recombinantendotoxin-neutralizing polypeptide according to claim 19, wherein thepolypeptide contains neutral or anionic residues at BPI amino acidresidue positions 27, 30, 33, 42, 44, 48, and
 59. 21. A recombinantendotoxin-neutralizing polypeptide according to claim 19, wherein thepolypeptide contains neutral or anionic residues at BPI amino acidresidue positions 77, 86, 90, 96, 118, and
 127. 22. A recombinantendotoxin-neutralizing polypeptide according to claim 19, wherein thepolypeptide contains neutral or anionic residues at BPI amino acidresidue positions 148, 150, 160, 161, 167, 169, 177, 185, and
 198. 23. Arecombinant endotoxin-neutralizing polypeptide according to claim 18,wherein the polypeptide contains neutral or anionic residues at BPIamino acid residue positions 27, 30, 33, 42, 44, 48, 59, 77, 86, 90, 96,118, 127, 148, 150, 160, 161, 167, 169, 177, 185, and
 198. 24. Arecombinant endotoxin-neutralizing polypeptide according to claim 14,wherein the polypeptide contains the amino acid sequence of LBP havingan amino acid substituted for an amino acid in a corresponding aminoacid residue position of BPI.
 25. A recombinant endotoxin-neutralizingpolypeptide according to claim 24, wherein the amino acid substituted isat LBP amino acid residue positions 77, 86, 96, 118, 126, 147, 148, 158,159, 161, 165, 167, 175, 183, or
 196. 26. A recombinantendotoxin-neutralizing polypeptide according to claim 14, wherein thepolypeptide contains the amino acid sequence of BPI having a cysteineresidue substituted with an amino acid other than cysteine.
 27. Arecombinant endotoxin-neutralizing polypeptide according to claim 26,wherein said cysteine residue is at BPI amino acid residue position 132,135, or
 175. 28. A recombinant endotoxin-neutralizing polypeptideaccording to claim 26, wherein the cysteine residues of BPI at positions132, 135, and 175 are substituted with an amino acid other thancysteine.
 29. A recombinant endotoxin-neutralizing polypeptide accordingto claim 14, wherein the polypeptide comprises a molecule which enhancesthe half-life of said polypeptide and is covalently bound to thepolypeptide.
 30. A recombinant endotoxin-neutralizing polypeptideaccording to claim 29, wherein said polypeptide contains alipopolysaccharide-binding domain of BPI, LBP, a BPI variant, or an LBPvariant.
 31. A recombinant endotoxin-neutralizing polypeptide accordingto claim 29, wherein said molecule is an immunoglobulin fragment, ahalf-life enhancing portion of LBP, a half-life enhancing portion of anLBP variant, or polyethylene glycol.
 32. A recombinantendotoxin-neutralizing polypeptide according to claim 29, wherein theendotoxin-neutralizing polypeptide of (a) is a C-terminal fragment ofBPI and the molecule of (b) is an N-terminal fragment of LBP.
 33. Arecombinant endotoxin-neutralizing polypeptide according to claim 32,wherein said C-terminal fragment of BPI is a fragment having an aminoacid sequence contained in BPI amino acid residues 60-456.
 34. Arecombinant endotoxin-neutralizing polypeptide according to claim 33,wherein said C-terminal fragment of BPI is BPI amino acid residues60-456, 136-456, 277-456, 300-456, 200-456, 136-361, 136-275, 200-275,or 200-361.
 35. A recombinant endotoxin-neutralizing polypeptideaccording to claim 32, wherein said N-terminal fragment of LBP is afragment having an amino acid sequence contained in LBP amino acidresidues 1-175.
 36. A recombinant endotoxin-neutralizing polypeptideaccording to claim 21, wherein said N-terminal fragment of LBP is LBPamino acid residues 1-59, 1-134, 1-164, 1-175, 1-274, 1-359, 1-134, or1-197.
 37. A recombinant endotoxin-neutralizing polypeptide of claim 18,wherein the polypeptide further comprises a C-terminal fragment of LBP.38. A recombinant endotoxin-neutralizing polypeptide of claim 23,wherein the C-terminal fragment of LBP is LBP amino acid residues360-456 or 274-456.
 39. An isolated DNA molecule encoding a recombinantendotoxin binding polypeptide according to claim
 14. 40. A vectorcomprising the DNA of claim
 39. 41. A transformed host cell comprisingthe DNA of claim
 39. 42. A method for producing a recombinantendotoxin-neutralizing polypeptide according to claim 14, said methodcomprising the steps of: culturing a transformed host cell comprisingDNA encoding a recombinant endotoxin binding polypeptide according toclaim 14, said DNA being operably linked to a promoter for expression ofthe polypeptide encoded by the DNA, said culturing being underconditions allowing expression of said polypeptide; and isolating therecombinant endotoxin binding polypeptide produced.
 43. A pharmaceuticalcomposition comprising: a therapeutically effective amount of arecombinant endotoxin-neutralizing polypeptide according to claim 14;and a pharmaceutically acceptable carrier.
 44. A method of treating asubject suffering from an endotoxin-related disorder, said methodcomprising: administering to a subject having an endotoxin-relateddisorder a therapeutically effective amount of a recombinantendotoxin-neutralizing polypeptide according to claim 14, whereinLPS-mediated stimulation of neutrophils and mononuclear cells isinhibited.
 45. A method of preventing an endotoxin-related disorder in asubject, said method comprising: administering to a subject aprophylactically effective amount of a recombinantendotoxin-neutralizing polypeptide according to claim 14, wherein theendotoxin-related disorder is prevented.